Genomic DNA encoding the prespore cell-specific PL3 cDNA was cloned and sequenced, revealing that the gene consists of three exons separated by short 100-bp introns. The single long open reading frame predicts a primary translation product of 70 kDa after removal of a cleavable signal peptide, two-thirds of which consists of four kinds of amino acid repeat elements, including two found in other spore coat proteins. The 85-kDa PL3 protein synthesized in vivo accumulates specifically in regulated secretory vesicles of prespore cells (prespore vesicles), as determined microscopically using antibody against a PL3 gene fusion product expressed in Escherichia coli. The protein later accumulates extracellularly in the spore coat, which is formed during sporulation, as determined ultrastructurally and by Western blot analysis of SDS-PAGE gels. In addition to its high proportion of repeat elements, the PL3 protein has the following properties which distinguish it from other spore coat proteins: (1) it is located at the outer extent of the middle layer, beneath the outer layer, (2) its dissociation from the coat requires the presence of protein denaturants and reducing agents at elevated temperature, and (3) a large proportion of the protein is not dissociated from the coat even under these conditions, as determined by ultrastructural analysis of the extracted coat. The PL3 protein may contribute to the structure of the coat at the interface between the middle, cellulosic layer and the outer, electron-dense, proteinaceous layer.