The enzyme cholesterol oxidase (E.C. 1.1.3.6), purified from a Streptomyces culture filtrate was found to have strong oral insecticidal activity on neonate larvae of the boll weevil (Anthonomus grandis grandis Boheman) from a laboratory population. The LC₅₀ and LC₉₅ against neonate boll weevil larvae in 14 day bioassays were 0.24 and 4.52 mg/mL, respectively. Cholesterol oxidase also showed moderate insecticidal activity against lepidopteran larvae. Larvae of several species were stunted severely by the enzyme at 100 mg/mL in 7 day assays. Extended feeding (27 days) by Heliothis virescens at this concentration resulted in 97% mortality with no survivors pupating. Cholesterol oxidase, cloned and expressed in tobacco plants, was bioactive as shown in bioassays with boll weevil larvae. These studies were conducted to further evaluate the utility of cholesterol oxidase in a program to establish boll weevil-resistant transgenic cotton.