Rb+ uptake, intracellular Na+ and K+ levels, and the tissue-medium distribution of the nonmetabolized glucose analog, 3-O-methyl-D-glucose (3-MG) were measured in rat diaphragms incubated with chlormadinone acetate, 6-chloro-4,6-pregnadien-17-ol-3,20-dione 17-acetate (CMA), in the presence and absence of ouabain. CMA in concentrations of 5 X 10(-7) M or higher significantly depressed 86Rb uptake, and promoted an increase in internal Na+ and a decrease in internal K+, indicating inhibition of the sodium pump. Sugar transport in resting muscle parallels the changes in internal Na+ levels and is an additional indicator of sodium pump activity. Equilibration of 3-MG between tissue and medium was accelerated by CMA, in parallel to the rise in internal Na+ level. Effects of CMA on Na+ levels and sugar transport, but not on Rb+ uptake, were additive to those of various concentrations of ouabain, suggesting interaction with sites not affected by ouabain. These results on diaphragm muscle confirm our previous studies on isolated cardiac muscle preparations showing that CMA, added to the aqueous bathing medium, inhibits the sodium pump in intact muscle tissues.