AbstractTetracosahexaenoic acid (24:6ω‐3) is an intermediate in the conversion of 18:3ω‐3 to 22:6ω‐3 in mammals. There is limited information about whether cells can assimilate and metabolize exogenous 24:6ω‐3. This study compared the effect of incubation with 24:6ω‐3 on the fatty acid composition of two related cell types, primary CD3⁺ T lymphocytes and Jurkat T cell leukemia, which differ in the integrity of the polyunsaturated fatty acid (PUFA) biosynthesis pathway. 24:6ω‐3 was only detected in either cell type when cells were incubated with 24:6ω‐3. Incubation with 24:6ω‐3 induced similar increments in the amount of 22:6ω‐3 in both cell types and modified the homeoviscous adaptations fatty acid composition induced by activation of T lymphocytes. The effect of incubation with 18:3ω‐3 compared to 24:6ω‐3 on the increment in 22:6ω‐3 was tested in Jurkat cells because primary T cells cannot convert 18:3ω‐3 to 22:6ω‐3. The increment in the 22:6ω‐3 content of Jurkat cells incubated with 24:6ω‐3 was 19.5‐fold greater than that of cells incubated with 18:3ω‐3. Acyl‐coA oxidase siRNA knockdown decreased the amount of 22:6ω‐3 and increased the amount of 24:6ω‐3 in Jurkat cells. These findings show exogenous 24:6ω‐3 can be incorporated into primary human T lymphocytes and Jurkat cells and induces changes in fatty acid composition consistent with its conversion to 22:6ω‐3 via a mechanism involving peroxisomal β‐oxidation that is regulated independently from the integrity of the upstream PUFA synthesis pathway. One further implication is that consuming 24:6ω‐3 may be an effective alternative means of achieving health benefits attributed to 20:5ω‐3 and 22:6ω‐3.