We have previously determined the dietary alpha-linolenic requirement for membrane synthesis in the developing animal. This study measures the dietary requirement for maintaining normal membrane composition in adult rats, as determined by 22:6(n-3) (docosahexaenoic acid) concentration. Sixty-day-old rats, previously fed a diet containing both linoleic and alpha-linolenic acid, were divided into nine groups, each receiving different quantities of alpha-linolenic acid but the same amount of linoleic acid. They were killed 4 wk after initiation of the new diet to determine the minimum quantity of alpha-linolenic acid required in the diet for maintaining the 22:6(n-3) tissue concentration in brain (whole tissue, myelin and nerve endings), liver and heart. The minimal amount of dietary a-linolenic acid that maintained the maximal 22:6(n-3) level and minimal 22: 5(n-6) level in tissues was considered to be the dietary requirement. The quantity was found to be 1.30 g/kg diet (0.26% of dietary energy). It was lower than that found for the developing animal (0.4% of energy). At lower quantities of dietary alpha-linolenic acid, 22:6(n-3) was replaced by 22:5(n-6) in the organs examined, except in nervous tissue, in which 22:6(n-3) was highly preserved.