Abstract Degenerate PCR primers were designed based on the published nucleotide sequences of the sporulation sigma factor ***oE (spoIIGB) from Bacillus subtilis, Bacillus thuringiensis, and Clostridium acetobutylicum. The primer set was used in a Hot Start Touch Down-PCR to screen for the presence of the target gene in both spore-forming (eight Bacillus species, eight Clostridium species, Paenibacillus polymyxa, Thermoanaerobacterium thermosaccharolyticum, Moorella thermoacetica) and in nonspore-forming bacteria. Under optimized PCR conditions, all spore-forming bacteria tested yielded a PCR product of the expected size (∼360bp), although the nonspore-forming Listeria monocytogenes and Lactococcus lactis subsp. lactis also yielded PCR products of this approximate size. To improve the specificity and sensitivity of this assay, we Southern blotted gel electrophoresis-separated PCR products with a digoxigenin-labeled B. subtilis spoIIGB probe. This probe hybridized with the ∼ 360 bp PCR product from all spore-forming species but did not hybridize with PCR products of this approximate size from any nonspore-forming bacteria. The PCR-Southern blot assay was 100 to 1,000-fold more sensitive than PCR alone, yielding a lower detection limit of approximately 3 CFU spore-forming bacteria/PCR reaction. We conclude that, based on amplicon size and Southern hybridization, this strategy provides a viable approach for detecting spore-forming bacteria.