Protamines are small arginine-rich proteins that package DNA in spermatozoa. The mouse protamine 1 (Prm-1) gene is transcribed exclusively in post-meiotic spermatids. To identify elements in the Prm-1 promoter required for spermatid-specific transcription, we generated transgenic mice by microinjection of transgenes containing Prm-1 5' flanking sequences with 5' truncations or internal deletions of conserved sequences linked to a marked Prm-1 gene. We also tested Prm-1 promoter regions with a heterologous human growth hormone reporter gene. We conclude that a 113-bp region can direct spermatid-specific transcription and we have defined sequences within this region that are essential for proper function. These results will facilitate the isolation and characterization of transcription factors essential for post-meiotic gene expression.