We studied the erythrocyte Na,K-pump in chronically hemodialyzed uremic patients, immediately before and after a 4-h period of hemodialysis. Using [3H]ouabain as a probe, the number of Na,K-pump units per erythrocyte did not differ in uremic and control subjects, and hemodialysis had no acute effect on this parameter. In contrast, in these same cells the mean level of Na,K-pump-mediated 86Rb transport was 30% lower in predialysis uremic patients than in controls, and this diminution in the rate of 86Rb transport per pump unit was improved after 4 h of hemodialysis in 17 of 18 subjects. The results of in vitro incubation of normal cells with pre- and post-dialysis sera from uremic patients suggested that a serum factor is responsible for the observed inhibition of Na,K-pump activity. Changes in cell Na concentration during dialysis did not appear to be responsible for the increased rate of Na,K-pump turnover after hemodialysis. However, there was a significant correlation between the extent of rise in pump-mediated 86Rb uptake and the weight loss that occurred during dialysis. We conclude that the ion transport turnover rate of the erythrocyte Na,K-pump is impaired in uremia by a nonouabain like circulating factor. This factor, whose activity is diminished acutely by hemodialysis, may play an important role in the systemic manifestations of the uremic syndrome, and could be an important endogenous regulator of the Na,K-ATPase.