Docosahexaenoic acid (DHA; 22:6n-3) is found in extremely high levels in human ejaculate with the majority occurring in the spermatozoa. However, the relative concentration of DHA and other fatty acids, in blood serum, seminal plasma, and spermatozoa of asthenozoospermic vs. normozoospermic individuals is not known. We analyzed the phospholipid fatty acid composition of blood serum, seminal plasma, and spermatozoa of normozoospermic men and asthenozoospermic men in order to determine if DHA levels, as well as the levels of other fatty acids, differed. The serum phospholipid DHA levels were similar in the two groups, suggesting similar intakes of dietary DHA. On the other hand, seminal plasma levels of DHA (3.0 vs. 3.7%) and total polyunsaturated fatty acids (PUFA) (11.8 vs. 13.5%) were significantly lower in asthenozoospermic vs. normozoospermic men, respectively, while 18:1 (19.0 vs. 16.8%) and monounsaturated fatty acids (MUFA) (24.2 vs. 21.7%) were significantly higher in the asthenozoospermic vs. the normozoospermic men. Spermatozoa from asthenozoospermic men had higher levels of 18:1, 20:0, 22:0, 22:1, and 24:0 than sperm from normozoospermic men, and lower levels of 18:0 and DHA (8.2 vs. 13.8%). Furthermore, total MUFA (19.3 vs. 16.5%) was higher and total PUFA (19.0 vs. 24.0%), n-3 fatty acids (9.3 vs. 14.6%), and the ratio of n-3 to n-6 fatty acids (1.0 vs. 1.6) were lower in the asthenozoospermic men. Therefore, in asthenozoospermic individuals, lower levels of DHA in the seminal plasma, but not in the blood serum, mimic the decreased concentrations of DHA in the spermatozoa. This suggests that the lower concentrations of spermatozoon DHA in these individuals are due not to dietary differences but to some type of metabolic difference in the asthenozoospermic men.