The prodigiosins are reddish-colored tripyrrole antibiotics biosynthesized by several microorganisms such as Serratia, Pseudomonas and Streptomyces. Streptomyces griseoviridis 2464-S5 produces prodigiosin R1 and roseophilin, a unique prodigiosin-related compound containing two pyrrole and one furan moieties, and carries the rph gene cluster involved in their biosynthesis. Prodigiosin R1 and roseophilin differ in the cyclization pattern. Twenty-one of 25 genes in the rph cluster are homologous to red genes in Streptomyces coelicolor A3(2), which produces undecylprodiginine and butyl-meta-cycloheptylprodiginine. In the rph cluster, rphG, rphG2, rphG3 and rphG4 showed sufficient homology to redG, a gene involved in cyclization of undecylprodiginine to butyl-metacycloheptylprodiginine. Thus, we attempted to analyze the functions of these rph genes using a heterologous expression system in S. coelicolor. S. coelicolor M511 ΔredG (a redG-disrupted mutant) transformed with an rphG-expressing plasmid produced two cyclized prodigiosins (1 and 2) in addition to undecylprodiginine (3), whereas strains expressing rphG2, rphG3 or rphG4 produced only 3. Compound 1 was identified as metacycloprodigiosin by NMR and MS, and compound 2 was a new prodigiosin determined as propyl-meta-cyclooctylprodiginine. Cytotoxicity assays using HeLa and HT1080 cells showed cyclization of the alkyl chain did not significantly affect cytotoxicity. Known prodigiosin cyclization genes redG and mcpG are involved in cyclization of undecylprodiginine to butyl-meta-cycloheptylprodiginine and metacycloprodigiosin, respectively. Among the four redG homologous genes in the rph cluster, rphG was identified as a prodigiosin cyclization gene based on the prodigiosins produced by the rphG-expressing strain. RphG is presumed to cyclize isotridecylprodiginine to prodigiosin R1, although no prodigiosin with an 11-membered carbocyclic ring has been isolated from S. griseoviridis 2464-S5. Neither rphG2, rphG3 nor rphG4 was able to cyclize undecylprodiginine.