The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.