The rat preprotachykinin (PPT) gene encoding the neuropeptides substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide gamma was isolated from a lambda Charon 4A genomic library. Two overlapping clones contained all of the exons present in beta-PPT, including some 7 and 9 kb 5′ and 3′ flanking sequence, respectively. The presence of 1 major and 2 minor transcription initiation sites was determined from primer extension and nuclease protection experiments. Analysis of the nucleotide sequence homology between the rat and bovine revealed the presence of highly conserved regions throughout the entire coding region and within the 5′ flanking sequences. Primer extension and nuclease protection experiments demonstrated that the primary transcript is differentially spliced primarily into gamma- and beta-PPT mRNA in all tissues examined in the adult rat where the gene is expressed. beta-PPT mRNA contains all of the exons, whereas gamma-PPT mRNA lacks exon 4, which encodes part of the N-terminus of NPK. The alpha-PPT mRNA, which lacks exon 6 (the sequence encoding NKA and processing sites), comprises about 1% of the total PPT mRNA. An RNA secondary structure model is proposed to account for these specific exon exclusion events in the RNA splicing process. These results are discussed with regard to the mechanisms regulating SP gene expression and the functional significance of differential RNA splicing in the rat.