We previously reported that the sperm of rhesus monkeys and humans uniquely contain large amounts of desmosterol not found in other tissues and have a high concentration of the highly polyunsaturated n-3 fatty acid, docosahexaenoic acid (22:6 n-3). However, the lipid composition of the testis, from which sperm originate, is unknown. During puberty, the testis undergoes remarkable morphological changes as testosterone levels rise and sperm production begins. We hypothesized that testicular maturation might also involve dramatic changes in lipid composition. Accordingly, we characterized the sterol and fatty acid composition of the testis of rhesus monkeys throughout the lifespan, from birth to old age. Although the cholesterol content in the testis remained relatively unchanged throughout life, the desmosterol content first decreased from 59 microg/g in infants to 6 microg/g in prepubertal monkeys, increased to 83 microg/g during puberty, and reached a plateau of 248 microg/g in the young adult, where it remained into old age. The polyunsaturated fatty acid composition of the testis also changed markedly. Docosahexaenoic acid (22:6 n-3) increased from 5.1% of total fatty acids in infants and juveniles to 18.1% in postpubertal young adults. Although some n-6 fatty acids, arachidonic (20:4 n-6) and linoleic (18:2 n-6), decreased from 16.0% and 10.0% in prepubertal juveniles, respectively, to 7.1% and 3.3% in young adults; dihomogamma-linolenic acid (20:3 n-6), the precursor of 1 series PGs, increased greatly from 1.8% to 10.3%. Similar changes occurred in both membrane and storage lipids (phospholipids and triglycerides), respectively. After puberty, the testicular fatty acid pattern remained stable into old age. Our data demonstrated that puberty is accompanied by substantial changes in the lipid composition of the primate testis. These changes suggest that desmosterol and both n-3 and n-6 polyunsaturated fatty acids may have important roles in sexual maturation.