When [1-14C]7,10,13,16,19-22:5 was incubated with microsomes, it was not desaturated to 4,7,10,13,16,19-22:6 by an acyl-CoA-dependent 4-desaturase. Subsequent studies with rat hepatocytes showed that 7,10,13,16, 19-22:5 was the precursor of 4,7,10,13,16,19-22:6, but the pathway proceeded through 24-carbon fatty acids. The implication of this finding is that 24-carbon acids, when produced in microsomes, must move to a site for partial beta-oxidation. Subsequent studies using a number of labeled acids showed that peroxisomes chain-shorten fatty acids, which then move back to the endoplasmic reticulum for esterification. The biosynthesis of both 22:5(n-6) and 22:6(n-3) thus requires the extensive movement of fatty acids between subcellular compartments.