Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cycle. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerevisiae SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein alpha subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an approximately 43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is localized primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE immunocomplexes that cannot phosphorylate histone H1, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.