The isoquinolinesulfonamide H-89, an inhibitor of cyclic AMP-dependent protein kinases (EC 184.108.40.206, cAPrK), inhibited the Ca2+-ATPase activity of cardiac and skeletal muscle sarcoplasmic reticulum (SR) with concentrations giving half-maximal inhibition of 8.1 +/- 1.3 and 7.2 +/- 0.9 micromol/L, respectively. The effect of H-89 on cardiac SR Ca2+-ATPase (EC 220.127.116.11) was the same irrespective of the presence or absence of inhibitors of cAPrK and furthermore, was not affected by a neutralising monoclonal antibody raised against phospholamban. Thus, the action of H-89 in inhibiting SR Ca2+-ATPase would not appear to be mediated by inhibition of cAPrK to reduce the phosphorylation state of phospholamban. In both cardiac and skeletal muscle SR, the inhibition by H-89 was noncompetitive with respect to ATP at a low concentration of ATP (<1 mmol/L) and of a mixed pattern at high concentrations of ATP. H-89 produced a decrease in affinity of the SR Ca2+ pump to Ca2+ with an increase in the Km for Ca from 0.52 +/- 0.01 to 0.94 +/- 0.03 micromol/L (P < 0.05) in cardiac SR and from 0.39 +/- 0.01 to 0.79 +/- 0.02 micromol/L (P < 0.05) in skeletal muscle SR. These results suggest that H-89 inhibits SR Ca2+-ATPase by a direct action on the SR Ca2+ pump to decrease its affinity to Ca2+. Such an action may contribute to the pharmacological effect of H-89.