We have previously shown that oral diethylstilbestrol (DES) lowers the high serum enzyme levels characteristic of Duchenne's muscular dystrophy (DMD). The present studies were undertaken to assess the effect of DES on the efflux of enzymes from isolated mouse skeletal muscle. Thirty-four male mice were used. Half received daily subcutaneous injections of 10 mug diethylstilbestrol-diphosphate (DES-DP) in saline for up to 3 wk and half daily saline injections. Left gastrocnemii were isolated from control and treated mice, and placed in separate incubation media at 37 degrees C. The efflux of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) from each was compared over a 4- to 5-hr period. In 15 of 18 there was a reduction in efflux of both enzymes from muscles isolated from DES-DP-treated mice. The greatest effect was noted during the second hour, when the mean efflux of each enzyme was reduced about 30%. Minor differences in muscle weight, water content, and enzyme activities did not explain the reduced efflux. These results suggest that DES has either reduced the permeability of the sarcolemma or enhanced the intracytoplasmic stability of these enzymes. This is the first drug reported to reduce the spontaneous enzyme efflux from isolated skeletal muscle. It remains to be established that a similar effect accounts for the reduction in serum enzyme levels when DES is administered to persons with DMD.