Studies were undertaken to characterize the mechanism of action of the gonadal steroids responsible for decreasing growth of Stuphylococcw, uureu8 in witro.Progesterone or testosterone at 20 pg/ml significantly increased the leakage of '*C activity from staphylococci pre-loaded with 14C-glucose. This enhancement of leakage was not detected with Gram negative micro-organisms. Hormonal diminution of total uptake of alanine was relatively independent of temperature and of the phase of culture. Anaerobiosis increased the steroidal diminution of alanine uptake c. 2-fold. Fractionation of staphylococci following exposure to various 14C-substrates in the presence of progesterone at 40 pg/ml did not reveal any distinctive influences on macromolecular syntheses. Entry of the labels into cellular pools, however, wm altered for 8 of the 10 substrates tested. Exchange experiments detailed the effects of steroids on the efflux of internal alanine and lysine. With progesterone a t 40 pgfml, alanine effluxed from the internal pool 3 times as fast as from the corresponding controls. The opposite effect occurred with lysine and progesterone depressed its exit rate. The stepwise removal of cellular constituents indicated a preferential binding of hormones to cell wall components. Using 14C-progesterone or 1Gdiethylstilbestro1, 24% and 29%, respectively, of the added hormone was f h d y bound to mucopeptide preparations, compared to 1-5% bound t o whole cells or isolated cell walls. We suggest that the hormones interfere with the integrated functioning of membrane-associated processes.