Pretreated poultry litter was used in batch cultures for the production of Bacillus thuringiensis (Bt)-based biopesticide of lepidoptera- and diptera-specific Cry1 and Cry2, diptera-specific Cry4Ba and Cry11Aa and coleoptera-specific Cry3Aa toxins by Bt subsp. kurstaki 81, subsp. israelensis HD500 and subsp. tenebrionis 3203, respectively. Bt kurstaki 81 showed improved growth and produced more toxin in this medium as compared to other subspecies. Base and acid hydrolysis were tested as the methods of substrate Pretreatment. The use of poultry litter pretreated with 2 N HCl yielded 94% more bioinsecticidal protein than 2 N NaOH-pretreated poultry litter when Bt kurstaki 81 was cultured. With appropriate pretreatment, poultry litter demonstrated potential as a valuable raw material for a low-cost complex medium to produce Bt-based biopesticides. (C) 2009 Elsevier Ltd. All rights reserved.