The aim of this study was to investigate the possibility of a relationship between plasma homocysteine (Hcy) and phospholipid FA (PUFA) in healthy Australian males. One hundred thirty six healthy male subjects aged 20-55 yr were recruited from the Melbourne metropolitan area. Each volunteer completed a semiquantitative food frequency questionnaire and gave a blood sample. Plasma Hcy concentrations were determined by an established HPLC method; the plasma phospholipid FA were determined by standard methods. Plasma Hcy concentration was significantly negatively correlated with plasma phospholipid concentration of the PUFA 20:5n-3 (r = -0.226, P = 0.009), 22:5n-3 (r= -0.182, P= 0.036), 22:6n-3 (r= -0.286, P= 0.001), total n-3 (r = -0.270, P = 0.002) and the ratio n-3/n-6 PUFA (r = -0.265, P = 0.002), and significantly positively correlated with 20:4n-6 (r= 0.180, P= 0.037). In the partial correlation analysis, after controlling for serum vitamin B12 and folate concentration, plasma Hcy was significantly negatively correlated with the plasma phospholipid concentration of 22:6n-3 (r = -0.205, P = 0.019), total n-3 (r = -0.182, P= 0.038) and the ratio n-3/n-6 PUFA (r= -0.174, P= 0.048). Evidence indicates that an increased concentration of n-3 PUFA in tissues has a beneficial effect on cardiovascular health. Our findings provide further evidence that increased consumption of dietary n-3 PUFA increases the concentration of n-3 PUFA in plasma phospholipid, which is associated with a protective effect on cardiovascular diseases and lower plasma Hcy levels. The mechanism that might explain the association between plasma 22:6n-3 and Hcy levels is not clear.