A 7B2 cDNA clone was isolated from a rat insulinoma cDNA library. The 1.1-kilobase (kb) cDNA insert contained 1 open reading frame of 630 nucleotides which encoded a 210-amino acid sequence. The deduced rat 7B2 precursor peptide of approximately 24 kDa would yield an approximately 21-kDa peptide upon cleavage of the signal sequence. The rat 7B2 nucleotide and amino acid sequences were highly homologous to those of mouse, human, pig, toad, and salmon. Northern analysis revealed 7B2 expression in rat pituitary and brain and in the PC12 and RIN5F cell lines. A DNA clone containing the 5' end of the rat 7B2 gene was isolated from a rat genomic DNA library. A 3.5-kb fragment isolated from this clone contained 1.5 kb of 5' nontranscribed DNA and 2 kb of the transcriptional unit. Nucleotide sequence and primer extension analyses revealed a TATA-like sequence approximately 25 basepairs up-stream of the transcription start site. Sequence analysis also revealed three putative cis-acting elements in the 5' flanking region. The second exon encoded the first 73 amino acids of the 7B2 prepeptide. Plasmids containing the 7B2 promoter fused with a reporter gene were transiently transfected into LAN-5 cells. Expression was evident only when the first intron was included with the 5' flanking sequences in the construct and only when cells were treated with forskolin or a phorbol ester. The rat 7B2 gene and cDNA will be valuable tools for the identification of factors that regulate 7B2 gene expression.