Genetic manipulation in the mosquito larvicidal organism Bacillus sphaericus has been hampered by a lack of effective molecular biological techniques and tools. Currently available vectors for Bacillus subtilis are inappropriate as they are selected with antibiotics to which B. sphaericus is naturally resistant. Construction of chimeras between the Staphylococcus aureus plasmid pE194 and the B. subtilis direct selection vector pBD214 permitted selection of plasmid transformants with erythromycin. As with pBD214, direct selection of recombinants is accomplished by insertional inactivation of the plasmid-borne thymidylate synthetase gene and subsequent selection with trimethoprim.