The Ca²⁺-binding S100A1 protein is specifically and highly expressed in cardiomyocytes where it interacts with ryanodine receptors, SERCA2a and phospholamban. It is supposed to be an important regulator of cardiac function. Field-stimulation of fluo3-AM loaded cardiomyocytes from WT and S100A1 KO mice indicated no significant difference in Ca²⁺-transient amplitude and their decay rate constants. SR Ca²⁺ content also was not different as estimated by Ca²⁺ transients during caffeine application. However, stimulating the b-adrenergic system by application of 1 mM isopoterenol (ISO) revealed that the increase in the amplitude of Ca²⁺-transients was less pronounced in KO mice (2.2-fold WT vs. 1.8-fold KO), while the acceleration of the decay was similar. The difference between cardiomyocytes from WT and KO mice also appeared in voltage-clamped cells. Ca²⁺-currents were smaller in WT than in KO (5.0±0.3 vs. 8.1±0.3 pA/pF, at 0 mV). ISO stimulation was again less pronounced in KO (1.9-fold WT vs. 1.2-fold KO). In contrast to AM-loaded cells, Ca²⁺-transients and SR Ca²⁺-content were larger in KO cells (1.4- and 1.3-fold, respectively). These results indicate that S100A1 may play an important role in PKA-dependent pathways, which lead to positive inotropy. In addition, knock-out of S100A1 may induce an adaptive phenotype resulting in upregulation of L-type Ca²⁺ current. Sponsored by SNF,SCRTN,SER.