From a total of 22 hypertriglyceridemic subjects tested, 14 subjects were selected on the basis of normal postheparin plasma lipoprotein lipase (LPL) levels and the presence of LPL inhibitory activity in their fasting plasma. The inhibitory activity was detected in both the lipoprotein fraction (d less than 1.25 g/ml) and the lipoprotein-deficient fraction (d greater than 1.25 g/ml). Correlational analyses of LPL inhibitory activity and apolipoprotein levels present in the lipoprotein fraction (d less than 1.25 g/ml) indicated that only apolipoprotein C-III (ApoC-III) was significantly correlated (r = 0.602, P less than 0.05) with the inhibition activity of the lipoprotein fraction. Furthermore, it was found that LPL-inhibitory activities of the plasma lipoprotein fraction and lipoprotein-deficient fraction were also correlated (r = 0.745, P less than 0.005), though the activity in the lipoprotein-deficient plasma was not related to the ApoC-III or apolipoprotein E levels. Additional correlational analyses indicated that the LPL levels in the postheparin plasma of these subjects were inversely related to the levels of plasma apolipoproteins C-II, C-III, and E. To explain some of these observations, we directly examined the in vitro effect of ApoC-III on LPL activity. The addition of ApoC-III-2 resulted in a decreased rate of lipolysis of human very low density lipoproteins by LPL. Kinetic analyses indicated that ApoC-III-2 was a noncompetitive inhibitor of LPL suggesting a direct interaction of the inhibitor with LPL. Results of these studies suggest that ApoC-III may represent a physiologic modulator of LPL activity levels and that the incidence of LPL inhibitory activity in the plasma of hypertriglyceridemic subjects is more common than previously recognized.