Aim: To examine the relationship between sperm DNA damage and sperm nuclear histone (H2B) staining. Methods: We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone (H2B) staining and sperm chromatin integrity (assessed by sperm chromatin structure assay and expressed using the percentage of (i) DNA fragmentation index [%DFI] and (ii) high DNA stainability [%HDS)]) were evaluated. Results: Histone H2B immunocytochemistry demonstrated two nuclear staining patterns: (i) focal punctate staining; and (ii) diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men (7.7%± 4.6%vs. 1.6%± 1.2%, respectively, P < 0.01). We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm %DFI (r= 0.63, P < 0.01) and sperm %HDS (r= 0.63, P < 0.01). Conclusion: The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.