Self-cloning experiments with a high-copy-number plasmid and Streptomyces griseus IFO13350 led to the cloning of a 11-kb DNA fragment that conferred yellow pigment production on the host. The cloned fragment contained a gene cluster for carotenoid biosynthesis, in which two polycistrons, crtE (encoding geranylgeranyl pyrophosphate synthase)-crtI (phytoene dehydrogenase)-crtB (phytoene synthase)-crtV (functionally unknown methyltransferase-like protein) and crtY (lycopene cyclase)-crtT (functionally unknown methyltransferase-like protein)-crtU (beta-carotene dehydrogenase), were present in a convergent way. Since strain IFO13350 produced no detectable amount of carotenoids, an increase in the copy number of the crt gene cluster led to production of carotenoids at a detectable level. Overexpression of the stress-responsive sigmaB-like protein CrtS from Streptomyces setonii also activated the cryptic crt genes in S. griseus and conferred pigmentation. A CrtS homologue (sigmaCrtS) in S. griseus, which was predicted by a computer-aided homology search, caused carotenogenesis to the same extent as CrtS of S. setonii, indicating that the two sigmaB-like proteins were functionally the same. Yellow pigment production by S. griseus containing crtS under the control of a strong promoter on a high-copy-number plasmid resulted from activation of transcription of the crt genes, because overexpression of sigmaCrtS in S. griseus led to transcriptional activation of the promoters in front of crtE and crtY. S1 nuclease mapping showed that crtS itself was transcribed at a low level under the laboratory conditions, which may account for undetectable production of carotenoids. The crt genes were suggested to locate very near one end of the linear chromosome, since they were completely deleted in mutant HH1 having large deletions at both ends. The gene organization of crt in S. griseus is similar to that in S. coelicolor A3(2) where the whole crt gene set is near one end of the chromosome.