Human spermatozoa were processed for determination of DNA fragmentation with use of an in situ diffusion assay, so that those cells containing DNA fragmentation produce extensive peripheral dissemination of DNA fragments after lysis in an agarose microgel. Quantification of specific protein staining confirmed that sperm cells without DNA fragmentation had almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal protein in a collapsed and condensed state. This result suggests that a modified nuclear protein matrix associates with fragmented sperm DNA.