Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore. The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized. The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores. This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure. Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane. Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B. subtilis chromosome. This is a region where there are no other known spo, ger, or PBP genes. In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis. In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5*. The two PBPs could be distinguished by immunoassays.