The maximum rubratoxin-B yield was obtained at pH 5.5,, and by increasing the initial pH to near neutrality the yield decreased for both yeast extract sucrose (YES) and Sabouraud dextrose yeast extract (SDYE) media, but the concentration of mycotoxin was higher in YES medium. The rubratoxin-B yield from Penicillium purpurogenium decreased with increasing gamma-irradiation, and at 1.0 kGy no mycotoxin was detected at any pH values. In both the unirradiated and irradiated P. purpurogeniium cultures, as the rubratoxin-B synthesis increased from 46 to 72 h, the lipid content decreased. The concentration (mmoles/g dry wt mycelium) of puridine nucleotides in the mycelium of P. purpurogenium during growth in YES and SDYE media may be a factor in rubratoxin-B synthesis. An elevated NADPH/ NADP ratio favours fatty acid synthesis whereas a depressed NADPH/NADP ratio favours mycotoxin formation. The gamma-irradiation played a role in the regulation of rubratoxin-B biosynthesis.