Commercial cotton cultivars (Gossypium hirsutum L.) were evaluated for aflatoxin production in 1974, 1975, and 1976 crop seasons. Bolls were inoculated at different levels of maturity with Aspergillus flavus Link. In 1974, the fungus produced aflatoxin in bolls of all 11 cultivars infected, but ‘Acala SJ‐1’ and ‘Acala 1517’ were better subtrates for the production of aflatoxin than ‘Delcott 277’, ‘Stoneville 213’, ‘Coker 711’, ‘Stoneville 751‐N’, or ‘Deltapine SR‐1’. In each of the 3 years, the fungus produced the greatest amount of aflatoxin in bolls of most cultivars inoculated at 30 days post‐anthesis, but less in 20‐day inoculated bolls and the least in 40‐day inoculated bolls. ‘Stripper 31’ was a notable exception in 1975 became A. flavus produced most aflatoxin in bolls of this cultivar inoculated 40 days post‐anthesis. In 1976, 20‐day and 30‐day post‐anthesis‐inoculated bolls were harvested 10, 15, 20, 25, and 30 days after inoculation. Aflatoxin was found in closed bolls 10 to 20 days after inoculation. The amount of aflatoxin increased with each subsequent time interval. The presence of aflatoxin in closed bolls demonstrated that bolls do not have to open to have toxin formation. A single pink bollworm [Pectinophora gossypiella (Saunders)] exit hole, which serves as the entrance hole for the fungus, allows enough oxygen penetration into the boll for aflatoxin formation.