Mapping studies have indicated that hundreds of Kruppel-type zinc-finger (ZNF)-containing genes are present within the genomes of mammals. One-third or more of these genes contain a second, highly conserved element termed the Kruppel-associated box (KRAB). In a recent study, several partial cDNA clones encoding distinct KRAB elements were mapped in mouse. One of these sequences, clone KRAB10, was assigned to mouse Chromosome (Chr) 9. We have isolated the complete coding portion of the KRAB10-containing gene and demonstrate that it is identical to Zfp54, one of several related ZNF genes that are located on mouse Chr 17 in the interval that is deleted in the tw18 mutation. Sequence analysis has shown that the KRAB A domain encoded by Zfp54 is highly similar to the comparable motif encoded by its nearest known neighbor, Zfp51. However, other portions of the related proteins, including their ZNF domains, are noticeably different in structure. Studies of Zfp54 expression revealed the presence of transcript in several adult tissues and in embryos throughout development; however, elevated levels of transcript were detected only in adult testis and in 7-day-old embryos. These data suggest that Zfp54 gene expression is under spatial and temporal control and may, therefore, play important roles in male germ cell and embryonic development.