The inotropic activity of amrinone and its effects on cyclic nucleotide levels in rabbit papillary muscles with normal and depressed contractile function have been compared. The effects of amrinone on the cyclic (c) AMP hydrolytic activity of cyclic nucleotide phosphodiesterase (PDE) isoenzymes were also examined. Amrinone (2.4 × 10−4-1.2 × 10−3 M) produced a relatively weak (maximal increase 11%) positive inotropic effect in papillary muscles stimulated at the near optimal stimulation frequency of 1 Hz. In contrast, large positive inotropic responses (maximal 138–200%) were obtained with amrinone in papillary muscles in which contractile force had been depressed by: (a) lowering stimulation frequency to 0.4 Hz, (b) reducing extracellular Ca2+ concentration from 2.5 × 10−3 M to 6.3 × 10−4 M, (c) prior addition of sodium pentobarbitone (6.5 × 10−4 M). The EC50 values for amrinone under conditions (a), (b), and (c) were 3.0 × 10−3, 2.6 × 10−3, and 2.8 × 10−3 M, respectively. Force-frequency curves in rabbit papillary muscles were compared at normal (2.5 ×10−3 M) and low (6.3 × 10−4 M) extracellular Ca2+ concentration. Contractions at low frequencies of stimulation (< 0.4 Hz) were less sensitive to removal of extracellular Ca2+ than higher stimulation rates indicating that in the former situation, recycling of intracellular Ca2+ is more important for maintaining contractile force. The positive inotropic effects of amrinone in normal and papillary muscles with depressed contractile force were associated with similar increases in both cAMP (1.6-2.5 fold) and cGMP (2.6-4.0 fold) levels, despite marked differences in the degree of inotropy in these muscles. The cGMP inhibited cAMP-PDE (PDE III) was selectively inhibited by amrinone (IC50 9.9 × 10−5 M), whilst the activities of Ca2+/calmodulin-stirnulated PDE, cGMP-stimulated PDE and cGMP-insensitive PDE were only weakly affected (IC50 > 2.5 × 10−4 M). The results show an involvement of cAMP in the mechanism underlying the positive inotropic action of amrinone in cardiac muscle with depressed contractile force. Furthermore, it is suggested that there may be an enhanced effectiveness of cAMP on intracellular Ca2+ mobilization in depressed cardiac muscle.