Effects of ouabain on [Ca++]i and on contractility was measured in quin2 and fura2 loaded cultured neonatal rat cardiac myocytes. Addition of ouabain (5 x 10(-8) to 5 x 10(-6) M) to cultured myocytes exposed to balanced buffered salt solution (BSS) caused a transient increase in [Ca++]i, followed by slow oscillations for about 10 min, and by an elevated steady state level of [Ca++]i thereafter. Concentrations of ouabain between 10(-7) and 5 x 10(-7) M caused an increase in the amplitude of systolic motion (ASM) whereas concentrations above 10(-6) caused a decrease in the ASM, an increase in the beating frequency and an upward shift of the base line, indicating impaired relaxation. When ouabain was added to cardiac myocytes exposed to Ca++-free BSS the increase in [Ca++]i was not observed, but only a transient decrease. To investigate the effect of [K+]o on the ouabain-induced changes in [Ca++]i, ouabain was added to cells exposed to BSS containing low K+ concentration (1 mM instead of 5 mM in balanced BSS). In this medium the increase in ASM by ouabain was similar to that in balanced BSS. Addition of ouabain caused a transient decrease in [Ca++]i. There was no initial increase in [Ca++]i and the steady state level of [Ca++]i was not elevated as compared with the same cells before the addition of ouabain. Similar results were observed in cells loaded with quin2 or with fura2. In view of these results the mechanism of action of ouabain on cardiac myocytes is discussed.