The effect of phenothiazines (trifluoperazine and chlorpromazine) on the activity of the sarcoplasmic reticulum Ca2+ pump was investigated. These drugs have a biphasic action on the ATPase activity. They inhibit the enzyme at high concentrations, but below 150 microM, they promote a significant stimulation of the ATP hydrolysis, which is accompanied by a slight increase of the Ca2+ transport. Leaking of the membrane occurs only at drug concentrations above 150 microM. The phenothiazine stimulatory effect was not found in the isolated enzyme whose activity was inhibited at all concentrations studied. These results indicate that low concentrations of phenothiazines uncouple the sarcoplasmic reticulum Ca2+ pump without disrupting the membrane and that the drug stimulatory effect on the ATPase is not due to a direct interaction with the enzyme. It is suggested that a coupling factor or a specific microenvironment surrounding the enzyme regulates the association between the Ca2+ transport and the ATP hydrolysis by sarcoplasmic reticulum of the skeletal muscle cell.