The fatty acid composition of cell membranes can be modified in cell culture. The role of different fatty acid families in modulating phagocytosis and oxidative burst is not clear and therefore the influence of 18-carbon polyunsaturated fatty acids (PUFA) on these processes was examined. The mouse monocyte/macrophage line P388D1 was cultured in medium supplemented with 2 or 20 µmol/l 18:2n–6 (linoleic acid; LA) or 18:3n–3 (α-linolenic acid; LNA) and fatty acid enrichment of the cells was tested after 8 days. The macrophages were activated with phorbol ester in order to promote oxidative burst and intracellular dihydrorhodamine oxidation was determined. To test phagocytosis capacity uptake of fluorescence-labeled <i>Escherichia coli</i> was determined. Activation of the transcription factor nuclear factor (NF)-kappaB was also determined. Cells grown in medium with 20 µmol/l LA contained 2- to 3-fold more n–6 PUFA including 4-fold more arachidonic acid. Cells grown in medium with 20 µmol/l LNA contained 4-fold more n–3 PUFA. Both LA and LNA enhanced phagocytosis and decreased oxidative burst, with little difference between the fatty acids. NF-kappaB activation at 1 h post-stimulation was not affected by adding LA or LNA to the culture medium. We conclude that the fatty acid composition of macrophages influences their ability to phagocytose and mount oxidative burst.