Influence of different factors such as pH, temperature, carbon and nitrogen source on production of asparaginase by Thermomyces lanuginosus, Talaromyces luteus and Rhizomucor pusillus was investigated. All three fungi under investigation preferred modified Czepek Dox (medium E) and 8 days incubation, temperature of 40-45˚C and pH 6.0–7.0 for maximum production of asparaginase. Production of asparaginase varied with the carbon and nitrogen source present in the medium. Asparaginase production by all the three fungi was adaptive in nature.