1. The onset of ouabain inhibition was quantified by analysis with an integrated rate equation from experiments in which the activity of Na,K-ATPase from guinea-pig myocardium had been altered with adenosine 5'-triphosphate (ATP, 0.3-9 mmoll-1) in the absence and presence of a detergent. 2. Under control conditions with increasing ouabain (0.1-100 mumoll-1) and ATP (0.3-1 mmoll-1) concentrations, inhibition developed faster. The acceleration by ouabain became less effective at saturating concentrations leading to a non-linear relationship between pseudo-first-order rate constants of inhibition and ouabain concentration. With a rise of ATP to 3 and 9 mmoll-1, i.e., near total Mg concentration (5 mmoll-1), inhibition was retarded presumably because the free concentrations of Mg and uncomplexed ATP changed. Varying the ATP concentration had little effect on ouabain potency at steady state; Hill coefficients were less than 1. 3. The detergent alamethicin (23 micrograms ml-1) neither interfered with Na,K-ATPase activity nor with inhibition at steady state but accelerated its onset. This supports a role for a lipid barrier in the development of inhibition. 4. While the reaction of low concentrations of ouabain with the receptors seemed to govern inhibition rate, with an increase in steroid concentration in the presence of alamethicin, ATP-dependent enzyme activity interfered with the onset of inhibition. The transition of the enzyme between ouabain-sensitive and ATP-hydrolytic conformations consequently causes the non-linear concentration-dependence of pseudo-first-order rate constants. As the Hill coefficient was less than 1, a reaction of ouabain with two receptors also could have contributed to the special concentration-dependence of inhibition rates.