To determine the genomic organization of the mouse cyclin D1 locus (Cyl-1), a series of cosmids and cDNAs were recovered by hybridization with a genomic probe representing the 5' end of the homologous human gene, CCND1. Primer extension indicated that transcripts originate from one of three adjacent nucleotides at a single start site. Two overlapping cDNA clones that essentially accounted for the complete sequence of the larger 4.0-kb Cyl-1 transcript were characterized. A combination of RNase protection and sequencing across intron-exon boundaries established that the gene is organized into five coding exons with a long 3' untranslated region. Repeated attempts to isolate clones corresponding to the minor 3.5-kb RNA were compromised by the presence of an internal poly(A) domain. However, hybridization with specific probes revealed that the minor transcript lacks approximately 800 nucleotides from the 3' end of the major transcript and may be generated by a novel mechanism or by RNA processing.