Oleic acid (OA) stimulates apolipoprotein B (apoB) secretion from HepG2 cells by protecting the nascent protein from rapid intracellular degradation. In contrast, the n-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid, have been shown to reduce apoB secretion by increasing its intracellular degradation in rat hepatocytes. We attempted to determine if OA and DHA have these opposite effects at the same point in the secretory pathway for apoB or if they act at different points in HepG2 cells. Unexpectedly, we found that when DHA (0.2 mmol/L) was incubated with HepG2 cells for 2 hours, it stimulated both triglyceride (TG) synthesis and apoB secretion significantly (the "stimulatory effect"). The stimulatory effect of DHA on apoB secretion was associated with decreased intracellular degradation of newly synthesized apoB. These acute effects of DHA on TG synthesis and apoB secretion paralleled those previously demonstrated with OA. After DHA was removed from the medium, however, both TG synthesis and apoB secretion rapidly decreased to a level that was significantly less than the control level (the "inhibitory effect"). At the same time, intracellular apoB degradation was significantly increased, and this degradation was efficiently prevented by proteasome inhibitors. Removal of DHA from the incubation resulted in inhibition of the incorporation of endogenous fatty acids into TG. In contrast, removal of OA from the media was not associated with any such inhibitory effect. The inhibitory effect of DHA on basal apoB secretion persisted at least 8 hours. These studies suggest that incubation of HepG2 cells with DHA has biphasic effects on TG synthesis and apoB secretion: an initial stimulation of TG synthesis is followed by inhibition of TG synthesis and increased apoB degradation. Although the stimulatory effect of DHA is apparent during short incubations of HepG2 cells, both effects would be expected to occur during long incubations, since fatty acid uptake by cells is rapid and efficient. Thus, long incubations of HepG2 cells with DHA could result in overall reduced apoB secretion compared with cells incubated in bovine serum albumin. If these findings are extrapolated to the in vivo situation, they can explain the ability of dietary n-3 fatty acids, which would be delivered to the liver intermittently, to reduce very low density lipoprotein secretion.