To compare the extent of sperm DNA damage with the degree of protamine deficiency in spermatozoa of normal and subfertile individuals, 30 semen samples from three groups of subfertile individuals (oligozoospermic, asthenozoospermic and oligoasthenozoospermic) and 14 samples from normal individuals were collected from men referred to the Fertility and Infertility Centre of Shariati Hospital, Tehran. DNA damage was measured using the alkaline Comet assay, and protamine deficiency was measured using chromomycin A3 (CMA3) staining. Results indicated a significant difference in the extent of DNA damage in spermatozoa of subfertile patients compared with normal patients (P < 0.01). Spermatozoa from oligoasthenozoospermic patients showed a higher level of DNA damage compared with the other two study groups of subfertile men. The percentage of CMA3-positive spermatozoa was also higher in subfertile individuals compared with normal patients (P < 0.01), with the highest level occurring in oligoasthenozoospermic patients. A direct correlation between protamine deficiency and sperm DNA damage was found for all subfertile patients studied.