Parotid secretory protein (PSP) is the most abundant gene product in the mouse parotid gland. In this tissue, its mRNA accumulates to approximately 5 X 10(4) molecules per cell. Our analysis of 11 mouse tissues indicates that the parotid secretory protein gene is expressed exclusively in the parotid gland. We have cloned a 27,000 base DNA segment of mouse chromosome 2 containing the PSP transcription unit. Hybridization with nascent parotid transcripts elongated in vitro indicates that the parotid secretory protein gene is the only gene present within the cloned DNA segment, which is expressed efficiently in the parotid gland. The gene is composed of eight introns and nine exons, whose sizes and order was determined by a novel S1 nuclease method. The parotid secretory protein transcription unit measures 8300 bases from cap nucleotide to poly(A) addition site. Using nascent parotid transcript hybridization, we localized the area of transcription termination to a 700 base-pair region located 2500 bases downstream from the poly(A) addition site. The initiating nucleotide of transcription was determined by primer extension sequencing of the PSP mRNA. The genomic DNA sequence upstream from the transcription initiation site includes both TATA and CAAT motifs. In addition, there are sequence elements homologous to the simian virus 40 and polyoma core enhancer sequences located at position -422 and -653, respectively.