DNase activity of Clostridium acetobutylicum was found to exhibit a relatively broad, acidic pH optimum of 3.5-4.5. The enzyme could be completely inhibited by addition of 0.2 M EDTA (final concentration), whereas heat treatment or addition of diethylpyrocarbonate proved to be unsuccessful. Activity measurements in various cell fractions indicated a localization of DNase at the outside of the cytoplasmic membrane. Maximal activity could be found in the stationary growth phase. With the acridine orange-DNA overlay method, three mutants (D-1, D-2, and D-1.1) could be isolated that were DNase-deficient. The mutation proved to be very stable (reversion frequency of 0.2%). All mutants were "leaky"; however, D-1.1 was less leaky than the other two.