The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured A7r5 smooth muscle were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. Upon stimulation with 5 microM vasopressin (AVP) a cytosolic Ca2+ gradient was observed whereby the highest increase was observed in the subplasmalemmal region. The maximal nuclear Ca2+ concentration ([Ca2+]n) attained a lower level than that in the cytoplasm ([Ca2+]c > [Ca2+]n). After the initial rise, a second sustained change of the Ca2+ level was found and the initial gradient ([Ca2+]c > [Ca2+]n) was preserved. In Ca(2+)-free solution containing 2 mM EGTA the maximal [Ca2+]c value after AVP stimulation was significantly lower than in the Ca(2+)-containing solution, but it remained higher than [Ca2+]n which was the same in both conditions. The initial Ca2+ rise was followed by a monoexponential decline. When the influx of Ca2+ through voltage-sensitive Ca2+ channels was blocked, the maximal and steady state values of [Ca2+]c but not of [Ca2+]n were lower as compared to the values in non-treated cells. Preincubation with 10 microM verapamil and 2 mM Ni2+ resulted in initial [Ca2+]c and [Ca2+]n rises which were not significantly different from the levels found in the absence of Ni2+, but the sustained phase was absent in both compartments. The differential effect with [Ca2+]c > [Ca2+]n was not observed if 1 nM AVP was applied instead of 5 microM AVP. The results indicate that cytosolic and nuclear Ca2+ stores behave differently with respect to their dependence on the agonist concentration and also with respect to the effect of Ca(2+)-entry mechanisms.