PCTAIRE 3 is a member of the PCTAIRE subfamily of cdc2-related serine/threonine protein kinases. In the present study, cDNAs encoding two isoforms of PCTAIRE 3 have been cloned and the genomic organization of the human PCTAIRE 3 gene is reported. The gene spans 28.15 kb on chromosome 1q31-32 and contains 16 exons. The major transcript of PCTAIRE 3, designated PCTAIRE 3a, has an open reading frame that is 474 amino acids in length. Transcripts for PCTAIRE 3a were evident throughout the brain and in the majority of tissues analyzed. A second transcript containing an insert that adds 90 nucleotides to the third exon of the gene was also identified. This transcript, designated PCTAIRE 3b, encodes a polypeptide of 504 amino acids. Expression of PCTAIRE 3b was limited to several subcortical nuclei of the basal gangli and the spinal cord and substantial levels of this transcript were not evident outside of the central nervous system. Primary sequence comparisons between different cdc2-related serine/threonine protein kinases reveal that these proteins are most heterogeneous in their N-terminal domains and the PCTAIRE subfamily is further diversified by the presence of isoforms within this region.