Environmental and nutritional factors play important roles in regulating production of mycotoxins. Few studies have been reported on the biosynthesis of patulin mycotoxin and the mechanisms that involve its biosynthesis in Penicillium expansum. Here, we investigated the effects of two culture media, pH, temperature, carbon and nitrogen sources and effect of carbon/nitrogen ratio on mycotoxin biosynthesis by P. expansum isolated from Moroccan cereals. It was found that pH and temperature had great influence on patulin production. Results also showed that carbon and nitrogen sources influ-enced patulin biosynthesis significantly in this strain. L-glutamate was optimized as important nitrogen source in synthetic culture medium. Effect of carbon/nitrogen ratio was evaluated which indicated the dependence of patulin production on this ratio. These results will provide useful information to better understand the regulatory mechanisms of patulin biosynthesis, and be helpful in developing effective means for controlling a mycotoxin contam-ination of foods and feeds.