The effects of the antimalarial agent, mefloquine, and its derivatives on Ca2+ uptake and release by crude microsomes from the rabbit skeletal muscle were investigated using a spectrophotometric method. These compounds diminished the rate of Ca2+ uptake and inhibited the Ca2+ pump ATPase activity of the microsomes. Except for quinine, they appear to have negligible effects on Ca2+ release channels. Of the compounds investigated, mefloquine had the most pronounced effect on Ca2+ uptake and was also the most potent (noncompetitive) inhibitor of Ca(2+)-ATPase (Ki: 53 microM). The ability of mefloquine to interfere with Ca2+ sequestration into the sarcoplasmic reticulum via inhibition of the Ca2+ pump ATPase, may explain some of its actions on the isolated skeletal muscle (relaxation, inhibition of twitch responses, diminution of caffeine contractures) observed in earlier studies. However, its contractile effects are less readily explained. The novel finding that mefloquine inhibits the Ca2+ pump ATPase of the skeletal muscle, suggests that it may have similar effects on the Ca(2+)-ATPases of other tissues.