AbstractWe previously characterized a nuclease‐hypersensitive fraction of mouse sperm chromatin, which is organized in a typical nucleosomal structure. A partial genomic library was constructed with the DNA from the nuclease‐hypersensitive chromatin, which revealed a high content in retroposon/retroviral DNA sequences. Here we report that the cloned nuclease‐hypersensitive DNA also contains clusters of potential sites for transcription factors: among those, binding sites for Oct‐1, Oct‐4, TBP, Ets‐1, and C/EBP are most abundant. This observation prompted us to ask whether mature spermatozoa contain the corresponding protein factors. Indirect immunofluorescence experiments show that all analyzed factors are indeed present in the sperm heads. Moreover, transcription factors are associated with the nuclease‐hypersensitive chromatin of spermatozoa, as endogenous nucleases that degrade the hypersensitive fraction also cause the concomitant release of transcription factors from sperm cells into the medium. Band‐shift assays with proteins extracted from the supernatant, and immunofluorescence analysis of sperm pellets, indicate that transcription factors are largely recovered in the supernatant while being absent or poorly retained in spermatozoa. The possible involvement of these factors in early embryogenesis is discussed. Mol. Reprod. Dev. 60: 97–106, 2001. © 2001 Wiley‐Liss, Inc.