Cottonseed storage protein (CSP) and several other proteins (bovine serum albumin [BSA], collagen and zein) stimulated aflatoxin production when incorporated into a defined medium containing sucrose and nitrate. With protein as the sole carbon and nitrogen source, collagen, but not BSA, CSP or zein, produced aflatoxin levels comparable to defined medium controls. A dose response study using CSP as the sole carbon and nitrogen source revealed that aflatoxin (r²=0.89, P<0.05) and biomass (r²=0.99, P<0.05) production were correlated with protein concentration. A pronounced stimulation of aflatoxin production was observed when utilizing medium containing CSP and sucrose, but without nitrate, in a 5-day fermentation. Cottonseed storage protein induced the production and secretion of an elastinolytic protease activity in A. flavus cultures, when no defined carbohydrate was present in the medium. In addition, inclusion of raffinose as a sole carbon source in the growth medium supported aflatoxin production. The results suggest that seed storage protein composition and storage saccharides may be important factors influencing aflatoxin contamination in cottonseed.