Abstract: The present investigation was conducted to generate catabolite activation mutants of Aspergillus flavus through UV mutagenesis. Mutants of A. flavus were generated by exposure of spores suspension to UV irradiation at a distance of 13 cm in dark from the centre of germicidal lamp (240 nm). Quantitatively, mannanase activity was determined using dinitrosalicylic acid method, while protein content was determined by Lowry method. Mannanase production by the mutants varied with time of exposure to UV irradiation. All the mutants except for mutant designated AFUV90 showed higher specific mannanase activity in comparison with the parent strain. The isolated mutants were screened for catabolite activation studies in the presence of different mannose and glycerol concentrations (1 and 1% w/v) as carbon sources. The supplementation of 0.1 and 1% (w/v) mannose in the fermentation media caused activation of mannanase biosynthesis in 100 and approximately 91% of the mutants, respectively. The inclusion of 0.1 and 1% (w/v) glycerol induced an improvement in approximately 82 and 55% of the mutants, respectively in terms of mannanase biosynthesis. The generation of catabolite activation mutants through UV mutagenesis might be considered as a break through in the industrial production of mannanase.