The gene encoding rat peptidylglycine alpha-amidating monooxygenase (PAM) contains 26 protein-coding exons. We identified two non-overlapping genomic clones encoding the 5' untranslated region (UTR) of the PAM gene. Exon 1 has 69 nucleotides flanked by perfect splice acceptor and donor sites, with a TATA motif 25 nucleotides upstream. Exon 0 lacks TATA or CAAT motifs and is embedded in a G + C-rich 800-nucleotide CpG island. The major products identified by RNase protection initiated in exon 0; only a minority of mRNAs initiated in exon 1. 5'-rapid amplification of cDNA ends (RACE) identified the same major transcriptional start sites in exon 0 in the atrium and neurointermediate pituitary. The 2.0-kb fragment upstream of exon 0 and the 1.3-kb fragment upstream of exon 1 were placed upstream of a luciferase-based reporter gene in both sense and antisense orientations. Expression of luciferase was observed in neuroendocrine and nonneuroendocrine cells with both sense constructs. A 0.2-kb fragment of the exon 0 PAM promoter containing multiple GC box elements supported expression of luciferase activity in all cell types. Expression of reporter genes in cells that do not normally express PAM suggests a need for more upstream or intronic information, a role for methylation, or a need for chromatin scaffolding for tissue-specific expression of the endogenous gene.