Antibodies were raised against purified germinationspecific cortex-lytic enzyme (GSLE) from spores of Bacillus megaterium KM which neutralized the ability of GSLE to germinate permeabilized spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE demonstrated that GSLE is spore-specific and that >90% of the GSLE is associated with the dormant spore cortex peptidoglycan as a phosphorylated 63 kD pro-form, which could only be visualized after lysozyme digestion of the peptidoglycan. During germination, the 63 kD pro-form of GSLE is processed to release the active enzyme, which had an apparent molecular weight of 30 kD. Inhibitor studies demonstrated that GSLE activation occurs as part of the commitment reaction and thus represents the first-identified enzymatic event to occur during germination triggering. Proteins that cross-react with anti-GSLE sera are present in spore fractions of other species.