The proacrosin gene is transcribed in diploid spermatogenic cells and translated in haploid round spermatids. In order to evaluate sequences which are involved in proacrosin gene transcription, DNA-protein interactions were analyzed in 1.2 kb of the 5'flanking region of the rat gene. 13 protein binding sites were identified by DNase I footprinting using nuclear extracts from rat testis and brain, respectively. Five footprints (F1, F3, F7, TS2, TS3) which suggest an interaction with testis specific nuclear factors were further examined by gel retardation assays. Three testis specific binding sites (F1, F7, TS2, located 472bp, 697bp and 1004bp upstream of ATG, respectively) could be identified with both methods. The binding site F1 contains a motif which is similar to a testis specific footprint found in mouse protamine 1 gene. The nucleotide sequence of F7 contains the recognition motif of an isoform of the transcription factor GATA1, which is expressed in testis. Furthermore F1 and F7 are located in that part of the 5'flanking region of the proacrosin gene, which can direct proacrosin gene expression in germ cells of male transgenic mice.