A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons with the corresponding sequences from numerous other species are presented. The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A-dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines. Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B. subtilis cells grown in the presence of excess uracil. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence. The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined. However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid.