Endogenous carbon monoxide (CO) is a by-product of heme breakdown. Recently some physiological functions of CO were revealed. However, mechanisms of its action on cardiac function remain unclear. Our group has shown that CO causes decrease of electrical and mechanical activity in mammalian myocardium (1), however, the ionic mechanisms of this effect was questionable. The aim of the present study was to investigate the effects of CO on ionic currents in isolated murine cardiomyocytes. Standard microelectrode technique was used to investigate the effects of CO (3×10-4M) on action potential duration (APD) in isolated electrically paced (6 Hz) ventricular preparations. The preparations were dissected from male outbred mice (20 g). Animals were euthanized by cervical dislocation before heart excision. The effects of CO on ionic currents in isolated ventricular cardiomyocytes were investigated using whole-cell patch clamp. Ventricular cardiomyocytes were enzymatically isolated from freshly excised hearts of male outbred mice. CO stock solution (10-3M) was prepared by bubbling Tyrode solution with 99% gaseous CO and used instantly before application to the experimental chamber. CO working solution (3×10-4M) was made by diluting the stock solution in standard Tyrode solution. CO (3×10-4M) caused significant (p(T)<0.05, n>6 for all groups) decrease of APD in murine ventricular preparation at 50 and 90% repolarization level (51.55±3.94% and 57.75±5.54%, respectively; values are means±S.E.M., compared by Wilcoxon test). Whole-cell patch clamp data indicated that 3×10-4M CO does not activate potassium delayed rectifier currents (Ito and IKur). On the contrary, it inhibited steady-state potassium current (10.98±2.24%). Also, CO significantly reduced peak calcium L-type current (ICaL) – by 26.78±2.58% of control peak amplitude measured at +10 mV. These results were confirmed by microelectrode experiments: in the presence of ICaL blocker nifedipine (10-6M), CO failed to induce any significant changes in electrical activity of murine ventricular myocardium preparations. It was previously shown that CO electrophysiological effects in myocardium can be mediated by cGMP-dependent signaling pathway (2). In accordance with this finding, in our whole-cell patch clamp experiments with GTP-free pipette solution CO did not affect ICaL. Thus, our data suggest that AP shortening in mammalian myocardium caused by CO is mediated mainly by its inhibitory effect on ICaL, which is putatively mediated by cGMP intracellular signaling pathway.