During spermiogenesis, the haploid phase of spermatogenesis, the genome is packaged into a highly compacted form and this process requires replacement of histones by protamines. In the mouse, protamines are encoded by two genes, which are transcriptionally regulated in testis. To understand the regulation of transcription of the mouse protamine 2 (mP2) gene, the tissue-distribution of sequence-specific interactions between nuclear proteins and promoter DNA sequences have been analyzed. Protein binding to the promoter region from 2370 to 165 was studied using DNase I footprinting and gel shift assays. Five protein binding sites were identified, which are recognized by nuclear proteins from either testis or liver. Site 1 from 264 to 248, contains part of a cAMP responsive element (CRE), which in testis is recognized by CREMt, an activator of post-meiotic transcription. Testicular protein(s) also binds to three other promoter domains: site 2, 287 to 267, a region containing a CAAT box, and sites 4 and 5, 2239 to 2210 and 2328 to 2311, sequences with similarity to consensus steroid hormone responsive elements (HRE). In contrast, interactions between the mP2 promoter and nuclear factors from liver, a tissue in which the mP2 gene is not transcribed, are observed at sites 1, 2, and 4, as well as at an additional region at site 3, 2202 to 2175. Because occupancy at site 3 appears to correlate with inactivation of the gene in non-testicular tissues, whereas testicular protein binding at site 5 appears to be associated with active transcription, we conclude that the mP2 promoter displays intricate tissue-specific patterns of protein/DNA interactions at key regulatory elements.