In a previous study, we demonstrated that the red blood cell Na+concentration and Na+, K+-ATPase activity are sex-dependent and race-dependent: a higher intracellular Na+concentration in blacks and men was associated with a lower Na+, K+-ATPase activity. To examine whether the low Na+, K+-ATPase activity is due to a decreased number of enzyme units, altered structure of the enzyme, or the presence of an endogenous digoxinlike substance, ouabain binding studies were performed on the same subject group. The measurements included displacement of [3H]Jouabain from its specific binding sites by unlabeled ouabain or potassium. The results demonstrate that groups with lower enzyme activity manifest lower numbers of total specific ouabain binding sites on the surface of the red blood cell (mean ± SD: blacks, 654 ± 24.4; whites, 806 ± 18.3; women, 806 ± 26.9; men, 728 ± 21.2). Other kinetic parameters of [3H]ouabain displacement appear to be the same among the groups. The respective red blood cell Na+and K+concentrations were negatively and positively correlated with the number of ouabain binding sites. Our findings suggest that the lower activity of red blood cell Na+, K+-ATPase in blacks and men is a function of a lower number of Na+-K+pump units. The results also indicate that sex and race should be considered when red blood cell ouabain binding is examined.