The effects of cyclopiazonic acid (CPA) were investigated on isolated skeletal muscle fibers of frog semitendinosus muscle. CPA (0.5-10 microM) enhanced isometric twitch but produced little change in resting tension. At higher concentrations (10-50 microM), CPA depressed twitch and induced sustained contracture without affecting resting and action potentials. In Triton-skinned fibers, CPA had no significant effect on myofibrillar Ca2+ sensitivity but decreased maximal activated force at concentrations > 5 microM. In intact cells loaded with the Ca2+ fluorescence indicator indo 1, CPA (2 microM) induced an increase in Ca(2+)-transient amplitude (10 +/- 2.5%), which was associated with an increase in time to peak and in the time constant of decay. Consequently, peak force was increased by 35 +/- 4%, and both time to peak and the time constant of relaxation were prolonged. It is concluded that CPA effects, at a concentration of up to 2 microM, were associated with specific inhibition of sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase in intact skeletal muscle and that inhibition of the pump directly affected the handling of intracellular Ca2+ and force production.