To assess the relationship between heparin and the associated increase in nonesterified fatty acids (NEFA) and their possible influence on NaK-ATPase during hemodialysis, we studied two groups of patients: (1) 12 patients on chronic hemodialysis dialysed with heparin and (2) 6 patients dialysed without heparin. All 12 patients who received heparin anticoagulation had a 7-fold rise in NEFA on average and also had an increase in circulating inhibitors of NaK-ATPase assayed by 3H-ouabain displacement from NaK-ATPase and/or by effect of plasma on the uptake of 86Rb by rat aortic rings. Serial assays in 3 of the patients receiving heparin showed NEFA and inhibitory changes to be at or near maximum within 30-60 min. Of the individual NEFA, the greatest relative increases were in oleic (18:1) and linoleic (18:2) acids, and the strongest correlations were between linoleic acid and both 3H-ouabain displacement (r = 0.94) and 86Rb uptake (r=0.86). However, a small and slower increase in NEFA also occurred in 3 of the patients dialysed without heparin. We conclude that heparin anticoagulation during dialysis leads to a rapid and marked increase in circulating NEFA, particularly the unsaturated fatty acids, with a corresponding interference with NaK-ATPase activity. The clinical significance of these findings is unknown. The rise in NEFA during dialysis without heparin in some patients suggests that factors other than heparin may also contribute to the rise in NEFA.