The cAMP-inducible prespore gene, PL3, encodes a protein which is a novel component of the spore coat. Unlike the well-characterized spore coat proteins (SP60, SP70, and SP96) which are found in the outer layer of the coat, the PL3 gene product is localized to a subregion of the coat beneath the outer proteinaceous layer. Moreover a substantial portion of the PL3 protein is tightly associated with the spore coat and not released under the conditions that led to the identification of the other coat proteins. The promoter for this novel spore coat gene is described. Unlike the other coat- protein gene promoters, it lacks the extensive CA-type elements. It contains two short CA boxes and five prominent G-rich regions. Sequential deletions from the 5' end of the promoter which remove both CA boxes as well as two of the G-rich regions reduce the level of expression but do not alter the spatial regulation of expression. Despite the sequence differences, the PL3 promoter still confers correct spatial, temporal, and cell type-specific regulation on a reporter gene. Escherichia coli β-galactosidase enzyme activity expressed under the control of this PL3 promoter first appears in randomly isolated cells at the loose mound stage. Because of the sensitivity of the assay, β-galactosidase activity is detectable prior to the appearance of the PL3 protein on Western blots and by immunofluorescence. Later the number of cells staining for β-galactosidase activity and the intensity of staining increases. During tipped mound, slug, and culminant stages, cells expressing β-galactosidase under the control of the PL3 promoter are localized to prespore regions and are spatially coincident with cells expressing the PL3 protein. © 1994 by Academic Press, Inc.