This study was designed to examine whether displacement of free fatty acids (FFA) from albumin (BSA) can explain the antilipolytic effect of chlorophenoxyisobutyrate (CPIB). Warfarin, which binds to albumin with equal affinity, was used to test the generality of this mechanism. The procedure was to measure the concentration of free drug needed to inhibit hormone-stimulated lipolysis in isolated rat epididymal fat cells, and the effect on this process of albumin, which binds both FFA ligand and drug. The free drug concentration was initially obtained by ultrafiltration studies with albumin and 14C-labeled CPIB or 14C-labeled warfarin in the absence of cells. When epinephrine-activated lipolysis was measured, inclusion of 0.3 mM albumin decreased the free CPIB concentration required for 50% inhibition from 1.8 mM (-BSA) to 0.08 mM. Warfarin also inhibited lipolysis more effectively in the presence of albumin, with 50% inhibition at 0.06 mM (+BSA) vs. 0.7 mM (-BSA). Both drugs showed a similar high-affinity binding constant to albumin of n = 1, k = 2--4 X 10(5) M-1, and both competitively displaced [14C]stearate, provided that a hydrophobic trap was present. The results are consistent with the possibility that the antilipolytic effect of CPIB and warfarin is mediated by way of a competitive displacement of FFA from albumin, or an analogous cellular binding site, with subsequent feedback inhibition of lipolysis.