In this study we report the isolation of mutants of Clostridium acetobutylicum ATCC 824 that are deficient in protease activity. The mutants were formed by use of Tn916 transposon mutagenesis, and this study further indicates this technique as a viable method for studying the genetics of C. acetobutylicum. The reduced protease phenotype can be distinguished on milk agar plates and was confirmed by enzyme activity measurements with the fluorescent protease substrate FITC-casein. The protease-deficient strains are capable of normal spore formation and solvent production.