As the precise role of nitric oxide (NO) as a modulator of myocardial contraction and the force-interval relationship remains unclear, the objective of this study was to examine the effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on baseline myocardial contraction, and the impact of both SNAP and the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on the force interval relation. Studies were performed using isolated rat papillary muscles. In the presence of basiline NOS blockade, nanomolar to micromolar concentrations of SNAP exerted a modest positive inotropic effect with a small but significant increase in twitch isometric tension (P < 0.007). Nanomolar concentrations of SNAP also reduced overall twitch duration (P < 0.007). These effects were not seen in control experiments using N-acetyl-penicillamine instead of SNAP. The force-frequency response (FFR) and post-rest contractile potentiation, mechanical correlates of sarcoplasmic reticulum (SR) Ca2+ handling, were also examined. Neither L-NAME nor SNAP had any effect on post-rest potentiation following rest intervals as long as 6 min, or on the negative FFR at stimulation frequencies between 0.3 to 1.7 Hz. However, L-NAME significantly blunted the net reduction in twitch duration between 0.3 Hz and 1.7 Hz compared to control (P = 0.006), an effect reversed by 100 nM SNAP. These results indicate that low concentrations of NO can modulate myocardial function by influencing myocardial inotropy and the time course of myofilament interaction, but do not impact significantly on the force-interval relation and, by inference, SR Ca2+ handling. Moreover, modulation of twitch duration occurs over a range of stimulation frequencies, suggesting a mechanistic role for NO in the changes in contraction and relaxation time intervals seen during changes in heart rate.