We have identified difference in protein-DNA interactions associated with the promoter of the mammalian spermatogenesis-specific Pgk-2 gene in expressing and nonexpressing cells, using a band shift assay. We compared DNA-binding activities in nuclear protein extracts from expressing adult testis cells versus nonexpressing prepuberal testis cells and nonexpressing somatic cells. One or two DNA-binding activities were found to be uniquely associated with the expressed state of Pgk-2, while a third appears to be associated with the nonexpressed state. All three of these activities map to a region within the first 40 bp upstream from the core promoter of this gene. The Pgk-2 core promoter lacks a TATA box but contain a GC box and a CAAT box. We show that the GC box binds the ubiquitous transcription factor Sp1 and that the CAAT box binds CTF-1, both of which are present in extracts from all three tissue types tested. These results suggest that tissue-specific transcription of the Pgk-2 gene is associated with changes in protein-DNA interactions occurring within a 40-bp enhancer region and that different arrays of protein-DNA interactions in this region are associated with the actively expressed state of the Pgk-2 gene in spermatocytes and spermatids and with the terminally repressed state of Pgk-2 in somatic cells.