Confluent skin fibroblasts from normal humans were incubated in serum free medium with up to 100 nmole/mL eicosapentaenoic acid (EPA; bound to albumin in a 4.6:1 ratio) and compared with cells incubated with oleic acid (OA) at similar concentrations. The rate of [14C]OA incorporation into triacylglycerol (TG) (nmol/mg/h) was approximately 5-fold greater than that of [14C]EPA. The mass of TG formed after incubation of fibroblasts with EPA was also significantly lower than that formed with OA (43.2 +/- 9.3 vs. 59.5 +/- 6.6 micrograms/mg cell protein, respectively, P = 0.006). The addition of excess, unlabeled EPA reduced the rate of incorporation of [14C]OA into TG whereas unlabeled OA stimulated incorporation of [14C]EPA into TG. When the cells were preincubated with human serum basic proteins (BP I, II and III), the mass of TG formed (compared to baseline) was significantly higher with the basic proteins whether OA or EPA was studied. Only BP I significantly stimulated the mass of cell phospholipids, an effect that occurred with either OA or EPA in the medium. The results suggest that in cultured normal human fibroblasts, OA is a better substrate for TG synthesis than EPA and that this effect may be accentuated by the presence of the basis proteins.