The mouse tryptophan hydroxylase gene was isolated and its intron/exon boundaries and putative regulatory sequences identified. To isolate the gene a mouse mastocytoma cDNA clone encoding tryptophan hydroxylase was used to identify and isolate ten overlapping DNA fragments from a mouse genomic library. Restriction mapping and sequence analysis of the clones revealed that the gene contains 11 exons and covers a region of DNA of approximately 21 kb. The transcription initiation site was mapped and the major site of initiation yields an untranslated leader sequence of 124 nucleotides. A minor initiation site is located 9 nucleotides 3' of the major site. The 5' untranslated sequence is interrupted by the first intron. Analysis of the sequence upstream of the initiation site showed the presence of several putative promoter and regulatory sequences. Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes.