The Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human RAD1 (hRAD1) and mouse RAD1 (mRAD1) homologs of the S. pombe Rad1 (Rad1) protein. The human RAD1 open reading frame (ORF) encodes a protein of 282 amino acids; the mRAD1 ORF codes for a protein of 280 amino acids. The human RAD1 and mRAD1 messengers are highly expressed in the testis as different mRNA species (varying from 1.0, 1.4, 1.5, to 3.0 kb). The hRAD1 and mRAD1 proteins are 30% identical and 56% similar to the S. pombe Rad1 protein. Sequence homology was also noted with the Saccharomyces cerevisiae Rad17p, the putative 3'-5' exonuclease Rec1 from Ustilago maydis, and the structurally related polypeptides from Arabidopsis thaliana and Caenorhabditis elegans. The degree of conservation between the mammalian RAD1 proteins and those of the other species is consistent with the evolutionary distance between the species, implicating that these proteins are most likely true counterparts. Together, this suggests that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionarily conserved between yeasts and higher eukaryotes. The human RAD1 gene could be localized on human chromosome 5p13, a region that has been implicated in the etiology of small cell lung carcinomas, squamous cell carcinomas, adenocarcinomas, and bladder cancer.