Human and mouse paralogues of the evolutionarily conserved mammalian HRAD9 and Mrad9 cell cycle checkpoint control genes have been isolated and called HRAD9B and Mrad9B, respectively. HRAD9B encodes a protein that is 414 amino acids long and is 55% similar and 35% identical to the HRAD9 gene product. The Mrad9B protein is 398 amino acids long and is 50% similar and 35% identical to its paralogue. We demonstrate that the encoded human protein is nuclear and can physically interact with checkpoint proteins HRAD1, HRAD9, HHUS1, and HHUS1B, much like HRAD9. Northern blot analysis to detect tissue specificity indicates that the human and mouse genes are expressed predominantly in the testis. The abundance of HRAD9B RNA, as judged by quantitative reverse transcription-PCR, is very low in most testicular tumors, particularly those of germ cell origin, i.e., seminomas, relative to normal testis control, nonseminomas, or Leydig tumor cells. RNA levels corresponding to HRAD17, another checkpoint control gene, demonstrated a similar pattern, but in general, higher quantities of this message were detected in samples. Furthermore, normal/tumor tissue differences were not as dramatic or consistent from sample to sample, especially for the seminomas. Our results demonstrate for the first time that HRAD9 and Mrad9 are part of a gene family and reveal a new genetic element encoding a product that interacts with multiple, known cell cycle checkpoint control proteins. The findings also indicate that HRAD9B can serve as a biomarker in particular for testicular seminomas and might be causally related to the disease.