We have determined the cDNA sequence and the gene structure of the human phosphorylase kinase beta subunit (PHKB). With 95% amino acid sequence identity, the predicted primary structure is highly similar to that of the rabbit beta subunit. At least 140 kilonucleotides in length, the gene is large and consists of 33 exons. Exons 26 and 27 are two homologous, mutually exclusively spliced exons in the middle of the gene, and exon 2 is a facultatively utilized cassette exon encoding an alternative N-terminus of the beta subunit. The previous assignment of the PHKB gene to chromosome 16 is confirmed by the successful screening of a chromosome 16-specific genomic library. Plaque hybridization at reduced stringency led to the isolation of two processed pseudogenes, PHKBP1 and PHKBP2, but of no other PHKB-related sequences.