The effect of action potential duration and elevated cytosolic sodium concentration on the force-frequency relationship in isolated rabbit, guinea pig and rat papillary muscle preparations was studied. Shortening of action potential duration in guinea pig and rabbit from 150-200 ms to values characteristic of rat (20-40 ms), using the K(ATP) channel activator levkromakalim (15 mumol.l-1), markedly reduced the force of contraction and converted the positive force-frequency relationship into negative one at longer pacing cycle lengths. This conversion was greatly enhanced in the presence of acetylstrophanthidin (0.2-1 mumol.l-1), an inhibitor of the Na-K pump. Acetylstrophanthidin (1 mumol.l-1) alone, however, had no effect on the force-frequency relationship. Prolongation of action potential duration in rat with inhibitors of cardiac K channels (4-aminopyridine [10 mmol.l-1] plus tetraethylammonium [2 mmol.l-1) increased the force of contraction and abolished the negative force-frequency relationship observed in rat at longer pacing-cycle lengths. It is concluded that both action potential duration and cytosolic sodium concentration are major determinants of the force-frequency relationship in mammalian myocardium.