The onset of inhibition of Na+,K(+)-ATPase from guinea-pig myocardium was quantified with pseudo-first-order rate constants in a series of 14 cardioactive steroids. From these data the association and dissociation rate constants of the steroid-receptor complex were calculated. It was then found that the association of the steroids with receptors but not the dissociation of the steroid-receptor complex determined the largely different inhibitory potencies. Consistent with this finding, at equieffective steroid concentrations the rates of inhibition varied only slightly. The correlation of the association rate with the hydrophobicity of the compounds suggests that hydrophobic interactions facilitate the access of the steroid to the receptor. A conformational transition of the vicinity of the receptor subsequent to the formation of the steroid-receptor complex seems to alter the hydrophobic properties of the receptor environment to make the dissociation rate independent from hydrophobicity.