We have studied the synthesis of docosahexaenoic acid (22:6 n-3) from linolenic acid (18:3 n-3) and eicosapentaenoic acid (20:5 n-3) in Y-79 human retinoblastoma cells and in a derived cell line, Y-79a, which has lost delta6-desaturase activity. Whereas Y-79 cells efficiently converted both radiolabeled 18:3 n-3 and 20:5 n-3 to 22:6 n-3, no synthesis of this fatty acid was observed in Y-79a cells from either 18:3 n-3 or 20:5 n-3. Y-79a cells elongated 18:3 n-3 to 20:3 n-3, which was further converted to 5,11,14,17-20:4 through a delta5-desaturation, elongated 20:5 n-3 to 22:5 n-3 and 24:5 n-3, and retained the ability for retroconversion. These results underscore the key role of delta6-desaturase in the synthesis of 22:6 n-3 and stress the importance of an adequate supply of this fatty acid in situations of reduced delta6-desaturase activity, such as in perinatal period, to prevent a 22:6 n-3 deficiency status.