Mimecan is a proteoglycan expressed by many connective tissues. It was originally isolated in a truncated form as a bone-associated glycoprotein, osteoglycin, and was considered an osteoinductive factor. Recently, we demonstrated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation. In this study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of continuous DNA sequence and contains eight exons. The newly discovered first exon, identified by 5'-rapid amplification of cDNA ends, consists of a 5'-untranslated region and is enriched in C+G nucleotides. Two transcription initiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively spliced RNA isoforms are generated by the use of two distinct splice acceptor sites in the third exon situated 278 base pairs apart. We determined a partial genomic structure of the human mimecan gene and demonstrated two alternatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functional importance. Using ribonuclease protection assay, we analyzed the patterns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.