We removed by recombinant deoxyribonucleic acid (DNA) techniques a small DNA segment from within a cloned gene (the 0.4 kb gene) in which transcription in under sporulation control in Bacillus subtilis. These deletion mutation was introduced into the B. subtilis chromosome by transformation with cloned DNA. Competent cells bearing a mutation (tms-26) that is closely linked to the 0.4 kb gene were transformed with linearized plasmid DNA containing the truncated 0.4 kb gene and the wild-type allele of the tms locus. Selection for Tms+ transformants yielded oligosporogenous mutants of unusually dark-brown colony pigmentation. This phenotype was caused by a mutation which mapped at or very near the site of the 0.4 kg gene deletion, whose presence and position in chromosomal DNA was confirmed by Southern hybridization analysis. Phase-contrast microscopy and electron microscopy showed that the mutation, which we designated as spoVG, impaired sporulation at about the fifth stage; bacteria harboring the spoVG mutation proceeded normally through stage IV of development but frequently lysed thereafter, apparently as a result of disintegration of an immature spore cortex. This identifies the 0.4 kb gene (or DNA in its immediate vicinity) as a new sporulation locus and shows that its product functions at a late stage in development.