A range of geometrical and positional isomers of 18-carbon acyl chains are potential components of diets containing fats from processed vegetable oils or ruminant animals. We have examined seventeen 18-carbon fatty acids, with cis or trans double bonds in positions 8-15, for their effects on desaturation and chain elongation of [1-14C]18:2(n-6) or [1-14C]18:3(n-3), primarily to 20:4(n-6) or 20:5(n-3) respectively, in cultured glioma cells. All trans monoenoic positional isomers inhibited delta 5 desaturation by approximately 60% when 18:2(n-6) was the substrate, with no positional discrimination evident; with 18:3(n-3) as substrate, only the 11- and 12-trans isomers were effective inhibitors of formation of 20:5(n-3). All cis positional monoene isomers, except for 12-cis 18:1, produced 25-30% inhibition of conversion of 18:2(n-6) to 20:4(n-6), but had little effect on the conversion of 18:3(n-3) to 20:5(n-3). For dienoic isomers, the presence of a 12-trans bond inhibited formation of both 20:4(n-6) and 20:5(n-3) and this was enhanced markedly when the other bond in the dienoic acid was 9-trans. Presence of a 9-trans or 15-trans bond with 12-cis gave little effect except for a slight inhibition of 20:4(n-6) formation by the 12-cis,15-trans 18:2 isomer. All-cis 20:3(n-9) blocked delta 5 desaturation, increasing 20:3(n-6) accumulation from 18:2(n-6) and suggesting that formation of 20:3(n-9) from 18:1(n-9) during essential fatty acid deficiency may further exacerbate the already compromised formation of 20:4(n-6). Further, the differential effects of various cis and trans isomers on the metabolism of 18:2(n-6) and 18:3(n-3) suggest that either delta 5 desaturation is not common in both pathways of conversion of 18:2(n-6) or 18:3(n-3) to their primary products or that selective interactions of trans and cis isomers occur when the essential fatty acid substrates are different.