The desaturation of [1-(14)C] 18:3n-3 to docosahexaenoic acid (DHA; 22:6n-3) is enhanced in an essential fatty acid deficient cell line (EPC-EFAD) in comparison with the parent cell line (EPC) from carp. In the present study, the effects of DHA on lipid and fatty acid compositions, and the metabolism of [1-(14)C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. DHA supplementation had only relatively minor effects on lipid content and lipid class compositions in both EPC and EPC-EFAD cells, but significantly increased the amount of DHA, 22:5n-3, eicosapentaenoic acid (EPA; 20:5n-3), total n-3 polyunsaturated fatty acids (PUFA), total PUFA and saturated fatty acids in total lipid and total polar lipid in both cell lines. Retroconversion of supplemental DHA to EPA was significantly greater in EPC cells. Monounsaturated fatty acids, n-9 and n-6PUFA were all decreased in total lipid and total polar lipid in both cell lines by DHA supplementation. The incorporation of [1-(14)C]18:3n-3 was greater into EPC-EFAD compared to EPC cells but DHA had no effect on the incorporation of [1-(14)C]18:3n-3 in either cell line. In contrast, the conversion of [1-(14)C]18:3n-3 to tetraenes, pentaenes and total desaturation products was similar in the two cell lines and was significantly reduced by DHA supplementation in both cell lines. However, the production of DHA from [1-(14)C]18:3n-3 was significantly greater in EPC-EFAD cells compared to EPC cells and, whereas DHA supplementation had no effect on the production of DHA from [1-(14)C]18:3n-3 in EPC cells, DHA supplementation significantly reduced the production of DHA from [1-(14)C] 18:3n-3 in EPC-EFAD cells. Greater production of DHA in EPC-EFAD cells could be a direct result of significantly lower levels of end-product DHA in these cells' lipids compared to EPC cells. Consistent with this, the suppression of DHA production upon DHA supplementation was associated with increased cellular and membrane DHA concentrations in EPC-EFAD cells. However, an increase in cellular DHA content to similar levels failed to suppress DHA production in DHA-supplemented EPC cells. A possible explanation is that greatly increased levels of EPA, derived from retroconversion of the added DHA, acts to offset the suppression of the pathway by DHA by stimulating conversion of EPA to DHA in DHA-supplemented EPC cells.