The present study investigated whether fatty acid compositions of testes are affected by the obese condition and dietary n-3 long-chain fatty acid (LCFA) intake. Male lean and obese Zucker rats were fed a 15 % (w/w, total diet) fat diet containing either 0 or 5·0 % (w/w, total fatty acids) n-3 LCFA for 8 weeks. Reproductive organ weights, sperm morphology and fatty acid composition of phosphatidylcholine (PC), and phosphatidylethanolamine (PE) of testes were analysed. The obese rats had significantly (P < 0·0001) smaller epididymides and seminal vesicles, larger prostates and abundant underdeveloped testes compared with lean rats. Diet treatment did not affect the sex organ weights. The effect of genotype on fatty acid composition was minor in PC and PE except for DHA (22 : 6n-3). The n-3 LCFA diet significantly (P < 0·0001) elevated 22 : 6n-3 and reduced arachidonic acid (20 : 4n-6) and DPA (22 : 5n-6) in testicular PC and PE of lean and obese rats compared with the control diet. The acylation of dietary n-3 LCFA into 22 : 6n-3 was 2-fold higher in obese rat testes than in lean rats fed the same diet. Underdeveloped testes had 70 % less 22 : 5n-6 in PC and PE than normal-size testes. Results indicate that testicular fatty acid composition is sensitive to dietary fat modulations and especially obese rats responded more to dietary n-3 LCFA than their lean counterparts. The selective reduction in 22 : 5n-6 in underdeveloped testes indicates that 22 : 5n-6 is important in male reproduction in rats and requires further study to define the role of elongation and desaturation in testicular development.