Background and Aims: Recently impaired activity of the Na⁺,K⁺-ATPase was found in pancreatic islets from rats depleted in long-chain polyunsaturated w3 fatty acids (w3-D rats). While the present study shows specifically the dynamics of carbamylcholine effects on cytosolic [Ca²⁺]_i in dispersed islet cells, ⁴⁵Ca efflux from prelabelled and perifused islets and insulin release from the same islets in w3-D rats. Methods: Cytosolic [Ca²⁺]_i in dispersed islet cells and both ⁴⁵Ca fractional outflow rate (FOR) and insulin output from islets prelabelled over 60 min preincubation at 8.3 mM D-glucose and then placed in a perifusion chamber were compared in control animals and w3-D rats. Results: Cytosolic free Ca²⁺ in dispersed islet cells was not significantly different in w3-D rats (165  4 nM; n = 451) and control animals (162  7 nM; n = 352) over 10 min perifusion in the presence of D-glucose (8.3 mM). The administration of carbamylcholine (0.1 mM) provoked a rapid increase in [Ca²⁺]_i, peaking within one minute at mean values of 327.4  45.6 and 324.7  19.7 nM in control and w3-D rats respectively. However shortly thereafter, the mean value for [Ca²⁺]_i became significantly higher in w3-D rats than in control animals, this persisting up to the end of the experiments (min 25), at which time the [Ca²⁺]_i averaged 208.0  19.8 nM (n = 97) in w3-D rats as distinct (p < 0.005) from 124.1  18.2 nM (n = 85). After 44 min perifusion in the sole presence of D-glucose (8.3 mM), the ⁴⁵Ca FOR from the prelabelled islets was comparable in w3-D rats (1.24  0.07 10^-2.min^-1) and control animals (1.33  0.07 10^-2.min^-1). Carbamylcholine (0.1 mM) provoked a rapid peak-shaped increase of Ca²⁺ FOR, the peak value being reached within 2-3 min. The increment above the paired value recorded at min 44 was not significantly different in control animals (n = 6) versusw3-D rats (n = 8), whether expressed in absolute terms (3.35  0.65 versus 5.19  0.11 10^-2.min^-1) or as the paired min 46-47/44 ratio (357  59 versus 465  103 %). The insulin output from the perifused islets exposed to 8.3 mM D-glucose was lower in w3-D rats than in control animals. In both cases, carbamylcholine provoked an early peak-shaped increase followed by a later reascension of secretory rate. The absolute value of the early increment in insulin release (nU.min^-1.islet^-1) was lower (p < 0.01) in w3-D rats (112.7  15.1; n = 7) than in control animals (208.9  24.7; n = 6); however, the relative magnitude as judged from the min 46/min 44 ratio was virtually identical in w3-D rats (143.3  3.5 %; n = 7) and in control animals (147.3  7.4%; n = 6). Moreover, the relative magnitude of the later increase in insulin release, as judged from the min 90/min 44 ratio, was higher (p < 0.005) in w3-D rats (153.1  7.6 %; n = 7) than in control animals (117.8  6.3 %; n = 6). Conclusion: The findings collected in dispersed islet cells are in fair agreement with prior observations indicating a greater relative increase in insulin out put in response to non-nutrient secretagogues in islets from w3-D rats incubated for 90 min at 8.3 mM D-glucose . These findings are compatible with an impaired activity of Na⁺,K⁺-ATPase in islet cells from w3-D rats. Moreover, the secretory data collected in perifused islets suggest an altered priming of insulin-producing cells during preincubation at 8.3 mM D-glucose, possibly attributable, in part at least, to the perturbation of hexose metabolism in the islets of w3-D rats.