The unsaturated fatty acid composition of phospholipids from different tissues frequently varies. Rat liver phospholipids contain esterified 22:6(n-3) while 22:5(n-6) is the major esterified 22-carbon acid in testes phospholipids. Both testes and liver synthesize polyunsaturated fatty acids. Microsomes, particularly from liver, have been used extensively to measure reaction rates as they relate to polyunsaturated fatty acid and phospholipid biosynthesis. None of these rate studies explain why specific acids are synthesized and subsequently esterified. In this study we compared the metabolism of [3-14C]-labeled (n-3) and (n-6) acids when injected via the tail vein, as a measure of hepatic metabolism, versus when they were injected directly into the testes. Liver preferentially metabolizes [3-14C]-labeled 24:5(n-3) and 24:6(n-3) to yield esterified 22:6(n-3), when compared with the conversion of [3-14C]-labeled 24:4(n-6) and 24:5(n-6) to yield 22:5(n-6). Both 24-carbon (n-3) acids were also converted to 22:5(n-3) but no labeled 22:4(n-6) was detected after injecting the two 24-carbon (n-6) acids. Differences in the hepatic metabolism of 24-carbon (n-3) and (n-6) acids to 22:6(n-3) and 22:5(n-6), versus their partial beta-oxidation to 22:5(n-3) and 22:4(n-6), are important in vivo controls. Surprisingly, in testes a higher percentage of radioactivity was found in esterified 22:6(n-3) versus 22:5(n-6) following injections, respectively, of [3-14C]-labeled 22:5(n-3) versus 22:4(n-6), which is the corresponding metabolic analog. Corresponding pairs of 24-carbon (n-3) and (n-6) acids, as they relate to metabolism, were processed in similar ways by testes. The relative absence of esterified 22-carbon (n-3) fatty acids, versus the abundance of 22- and 24-carbon (n-6) acids in testes phospholipids, does not appear per se to be due to differences in the ability of testes to metabolize (n-3) and (n-6) fatty acids. It remains to be determined if there is selective uptake of specific fatty acids by testes for use as precursors to synthesize polyunsaturated fatty acids.