Free fatty acids (FFA) released during the lipolysis of triglyceride (TG)- rich lipoproteins in vivo are generally believed to be bound to serum albumin. When hypertriglyceridemic (HTG) sera were lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL), there was an 11- to 18-fold increase in serum FFA levels, and a major portion (> 80%) of the FFA in serum was partitioned to lipoprotein fractions. The greatest portion (33%) of FFA in lipolyzed HTG serum was associated with newly formed flocculent remnants that banded just below low density lipoproteins (LDL) in the density gradient tube. Very low density lipoprotein (VLDL), LDL, and high density lipoprotein (HDL) fractions in lipolyzed HTG serum contained 18- to 29-times more FFA molecules than those in prelipolysis serum. Analysis of the fatty acyl chain composition of FFA in lipolyzed HTG serum showed that the extent of partitioning of saturated FFA into the lipoprotein fractions relative to that of polyunsaturated FFA was about 4.5- to 11-times greater than that partitioned into the free protein fraction; most (84%) of FFA partitioned into flocculent remnants were saturated fatty acids. In vivo lipolysis of TG- rich lipoproteins in HTG subjects, induced by heparinization, resulted in only a small (2.8-fold) increase in serum FFA and little or no increase in the partitioning of FFA to lipoproteins. However, in vitro incubation of the postheparin serum at 37°C for 90 min resulted in a 2.9-to 6.8-fold increase in the serum FFA level and the partitioning of > 66% of total serum FFA into lipoprotein fractions. Studies of the interaction of various plasma fractions from control and in vitro lipolyzed HTG serum with cultured mouse peritoneal macrophages (MPM) showed that FFA partitioned to lipoprotein fractions were highly cytotoxic to cultured MPM, whereas FFA partitioned to albumin at a 10 x greater concentration were not cytotoxic. The cytotoxic potencies of FFA bound to lipoproteins and albumin were further compared after in vitro incorporation of FFA (oleic acids) into LDL and to albumin. FFA bound to LDL but not to albumin were cytotoxic to cultured MPM; the cytotoxicity of FFA bound to LDL was more closely related to the FFA to LDL-cholesterol molar ratio than to the total FFA concentration the culture dish. The ability of FFA bound to LDL and albumin to induce foam cell formation was studied in THP-1 monocyte-derived macrophages, which were less susceptible to cytotoxicity produced by FFA hound to LDL than MPM. With a sublethal dose, FFA bound to LDL induced a significantly greater accumulation of cellular TG than did the same amount of FFA bound to albumin. The data from this study suggest that the partitioning of an excess amount of FFA to lipoproteins, which could occur locally at the site of lipolysis in vivo, may be atherogenic.