The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P₄₆) removes an ~15-kDa C-terminal domain generating a 30-kDa species (P₃₀) which is stable against further digestion. While P₃₀ is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P₄₆ to the active 41-kDa form of GPR (P₄₁). Trypsin treatment of P₄₁ cleaves the same bond in the C-terminal part of the protein as is cleaved in the P₄₆P₃₀ conversion. While the ~29-kDa species generated by trypsin treatment of P₄₁ is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P₄₁ which is 13°C lower than that for P₄₆ and the unfolding of P₄₁ at significantly lower concentrations of guanidine hydrochloride than for P₄₆, are further evidence for a difference in tertiary structure between P₄₆ and P₄₁, with P₄₆ presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P₄₆ and P₄₁. The removal of ~30% of P₄₆ or P₄₁ without significant loss in enzyme activity localized GPR's catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P₄₁ by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.