The genes encoding the six pyrimidine biosynthesis enzymes from the thermophile Bacillus caldolyticus were characterized by cloning and complementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome, the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-pyrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr operon of Bacillus subtilis. Different parts of the B. caldolyticus cluster were cloned in two orientations in the expression shuttle vector pHPS9. Complementation studies in B. subtilis established that expression of the pyr genes was dependent on the vector-borne promoter, suggesting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase (pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosphoribosyltransferase (pyrE) levels in cells grown under different conditions indicated that expression of the operon is repressed 7–9-fold by addition of uracil to the growth medium. Based on the nucleotide sequence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.