We previously reported that oleic acid (OA) rapidly increased apolipoprotein (apo) B secretion by suppressing early intracellular degradation of nascent apo B in Hep G2 cells and suggested that the suppression of apo B degradation is associated with triglyceride (TG) biosynthesis from OA. To determine whether the inhibition of apo B degradation is associated with increased TG synthesis or is a direct effect of OA, we examined the effect of another fatty acid, eicosapentoenoic acid (EPA), on apo B kinetics in Hep G2 cells, since it is well known to have hypolipidemic action in clinical studies. The incorporation of [3H]glycerol into cellular TG was stimulated five-fold when Hep G2 cells were incubated for 2 h with EPA or OA (0.4 or 0.8 mM-1.5% bovine serum albumin (BSA) complex). The incorporation of [14C]acetic acid into cellular cholesteryl ester (CE) was significantly decreased by EPA treatment, whereas OA did not affect CE synthesis. Similar effects of these fatty acids on cellular lipid synthesis were observed in long-term incubation (24 h). Apo B was linearly secreted into the medium during 3 h, and EPA and OA doubled the rate of secretion. In long-term (24 h) incubations, both fatty acids significantly increased the incorporation of [3H]leucine into secreted apo B radioactivity or the accumulation of apo B mass in the medium. Pulse-chase studies revealed that both EPA and OA reduced intracellular apo B degradation to a similar degree. The inhibition of apo B degradation was also observed when the cells were preincubated with either EPA or OA for 24 h. These results suggest that increased TG synthesis leads to suppression of intracellular apo B degradation, which is independent of the source of exogenous fatty acid.