Promoter-cloning plasmids were constructed and have been used to isolate transcriptionally active DNA fragments from Staphylococcus aureus. The plasmids contain a chloramphenicol acetyltransferase (CAT) gene of Gram-positive (G+) origin which lacks both its promoter and the sequence responsible for CAT inducibility. The ability of S. aureus promoters to direct CAT expression in Escherichia coli and Bacillus subtilis was examined. Two classes of staphylococcal promoter sequences have been obtained. Class I DNA fragments direct CAT expression in S. aureus, B. subtilis, and E. coli, while class II DNA sequences direct CAT expression only in the G+ hosts.