Background and Aims: Attention was recently drawn to the impaired activity of the Na⁺,K⁺-ATPase in pancreatic islets from rats depleted in long-chain polyunsaturated w3 fatty acids (w3-D rats). The present study deals with the dynamics of ouabain 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: Over 10 min perifusion in the sole presence of D-glucose (8.3 mM), [Ca²⁺]_i was not significantly different in w3-D rats (165  4 nM) and control animals (163  7 nM). The initial increase in [Ca²⁺]_i caused by ouabain (1.0 mM) occurred earlier and was of greater magnitude in control animals than in w3-D rats. Indeed, within 36 s, a peak value of 292  23 nM was reached in the former animals, whilst [Ca²⁺]_i remained close to basal value (169  7 nM) at the same time in w3-D rats. Thereafter, a multiphasic pattern prevailed in control animals, with a nadir value of 229  12 nM about 10 min after introduction of ouabain and a later reascension to reach 283  20 nM at min 25. In the w3-D rats, however, [Ca²⁺]_i slowly increased from 224  10 nM after one min exposure to ouabain to 304  28 nM at the end of the experiments (min 25). After 44 min perifusion in the sole presence of D-glucose (8.3 mM), the ⁴⁵Ca FOR 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). Ouabain provoked a monophasic increase in both ⁴⁵Ca efflux and insulin release. The increase in ⁴⁵Ca FOR was more rapid in control animals, averaging within one min + 0.47  0.18 10^-2.min^-1 in control rats, versus only 0.07  0.08 10^-2.min^-1 in w3-D rats. In the control rats, the insulin output averaged 460  22 nU.min^-1 per islet after 44 min exposure to D-glucose and increased by 103  32 nU.min^-1 within one min of exposure to ouabain. In the w3-D rats, however, the corresponding values were 269  27 nU.min^-1 and 34  23 nU.min^-1. Conclusion: 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.