Recent evidence indicates that a member of the Y box-binding family of transcriptional regulators is identical to p56, a predominant protein of messenger ribonucleoprotein complexes. The p56 protein is highly enriched in oocytes and testis, and a functional RNA binding mouse cytoplasmic homologue has been cloned and partially characterized. Because few potential testis-specific transcriptional regulators have been identified, the testis-enriched Y box-binding proteins represent trans-acting elements of a unique model system for the study of haploid gene expression. The 5' flanking region of the testis-specific, haploid-expressed mouse protamine 2 gene contains an element with a 9-of-12 nucleotide identity with the previously defined Y box consensus sequence. We have investigated the possible role of Y box-binding proteins in transcriptional regulation of protamine 2 using specific antibodies and DNA-protein binding assays. Western blot analyses with two different anti-p54/p56 antibodies demonstrate that a mouse homologue of Xenopus p54/p56 is present in transcriptionally active mouse testis nuclear extracts. Our results further indicate that the Xenopus Y box-binding proteins bind to an element 5' to the mouse protamine 2 gene. Similarly, binding of the mouse testis homologue to the protamine 2 Y box element is demonstrated by gel mobility shift and antibody supershift analyses. The demonstrated interactions between testis-enriched Y box-binding proteins and protamine 2 transcriptional control elements therefore represent a unique system for functional studies to determine the mechanism of regulation of haploid gene expression.