Diacetyl monoxime (DAM) is a negative inotropic agent. To identify the mechanism of its actions, electrical and mechanical studies with various cardiac tissues were carried out. DAM (0.2-20 mM) inhibited the contractile force in both normal and 22 mM KCl-depolarized (in presence of 10(-6) M isoproterenol) guinea-pig papillary muscles in a concentration-dependent manner. In general, there was a lack of major effects of DAM on sarcolemmal electrical properties. The fast action potentials were somewhat depressed and the slow action potentials were slightly enhanced. In chemically skinned pig ventricular muscles, the myofibrillar contraction induced in 6.25 pCa was inhibited by DAM in a similar concentration range. DAM also produced an apparent decrease in sensitivity toward Ca++ in this preparation. Myofibrillar adenosine triphosphatase assay showed similar results as in the skinned muscles. All DAM effects were reversible upon washout and could be partially antagonized by raising [Ca++]. Taken together, the negative inotropic effect of DAM cannot be ascribed to an inhibitory effect on the slow inward current, as suggested previously. An inhibitory effect at the myofibril level is a distinct possibility. Additional effects of DAM on the sarcoplasmic reticulum cannot be ruled out.