When 5 X 10(6) hepatocytes were incubated for 40 min with from 0.15 to 0.60 mM [1-14C]linoleic acid, [1-14C]6,9,12-octadecatrienoic acid, or [1-14C]8,11,14-eicosatrienoic acid, there was a concentration-dependent acylation of radioactive metabolites into both triglycerides and phospholipids. When the concentration of either [1-14C]linoleic acid or [1-14C]8,11,14-eicosatrienoic acid exceeded 0.3 mM, there was no further increase in the metabolism of either fatty acid to other (n-6) metabolites. When the concentration of [1-14C]6,9,12-octadecatrienoic acid exceeded 0.15 mM, there was an apparent substrate-induced inhibition in its metabolism to 8,11,14-eicosatrienoic acid. With all three substrates (0.3 mM), there was time-dependent metabolism to other (n-6) acids. Cells then were incubated simultaneously with 0.3 mM [1-14C]linoleic acid along with 0.15 to 0.45 mM 6,9,12-octadecatrienoic acid or 8,11,14-eicosatrienoic acid. These exogenous nonradioactive (n-6) acids suppressed but did not abolish the conversion of [1-14C]linoleate to radioactive arachidonate. These findings suggest that some linoleate is converted to arachidonate without intracellular mixing of 6,8,12-octadecatrienoic or 8,11,14-eicosatrienoic acids. This hypothesis is supported by the finding that exogenous linoleate did not markedly affect the metabolism of [1-14C]6,9,12-octadecatrienoic or [1-14C]8,11,14-eicosatrienoic acid by microsomal chain elongating or desaturating enzymes.