Two cDNAs that encode a protein with 87% identity to human cyclin B1 and that differ only in the length of their 3'-untranslated regions have been isolated from a 70Z/3B murine pre-B leukemia cell library. Three sizes of RNA transcripts were detected in Northern hybridization analyses of a variety of normal tissues and transformed cell lines using the cDNA inserts as probes. The expression of these RNAs can be modulated in tissue culture cell lines by physiologically relevant stimuli, increasing when cells are stimulated to proliferate and decreasing when cells are induced to differentiate. Moreover, RNAs from tissues that contain few proliferating cells have no detectable hybridizing transcripts. The coordinate regulation of these RNAs with other genes that are activated during the cell division cycle and the profound similarity of the predicted amino acid sequence to those of published cyclin B homologues indicate that these genes encode a murine cyclin B1. In Southern hybridization analysis of BALB/cAnPt genomic DNA digested with EcoRI, 12 fragments hybridized with the cDNA probes. Through Southern blot analyses of DNA from backcross and cogenic mice, recombinant inbred strains, and somatic cell hybrids, the genetic loci that produce the cyclin B1-related sequences (designated loci Cycb1-rs1 to Cycb1-rs9) were mapped on mouse chromosomes 5, 1, 17, 4, 14, 13, 7, X, and 8, respectively. Cycb1-rs6 (on chromosome 13) is discussed as the most likely candidate for an expressed structural gene locus.