Access to a membrane-impermeant biotinylation reagent as well as protease sensitivity was used to determine germination proteins' topology in the inner membrane (IM) of decoated dormant spores and intact germinated Bacillus subtilis spores. The proteins examined were four nutrient germinant receptor (GR) subunits, the GerD protein, essential for normal GR-dependent spore germination, the SpoVAD protein, essential for dipicolinic acid movement across the IM, the SleB cortex-lytic enzyme, and the YpeB protein, essential for SleB assembly in spores, as well as green fluorescent protein (GFP) in the spore core. GerD and SpoVAD as well as GFP in the spore were not biotinylated in decoated dormant spores. However, GR subunits, SleB, and YpeB were biotinylated 4 to 36% in decoated dormant spores, although these levels were not increased by higher biotinylation reagent concentrations or longer reaction times. In contrast, the germination proteins were largely biotinylated in germinated spores, although GFP was not. All of the germination proteins in the germinated spore's IM, but not spore core GFP, were largely sensitive to an exogenous protease. These results, coupled with predicted or experimentally determined structural data, indicate that (i) these germination proteins are at least partially and in some cases completely on the outer surface of the spore's IM and (ii) there is significant reorganization of these germination proteins' structure or environment in the IM during spore germination.