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A new gene encoding a 35.8-kDa mosquitocidal toxin (Mtx3; 326 amino acids) was isolated from Bacillus sphaericus SSII-1 DNA. Mtx3 is a new type of mosquitocidal toxin with homology to the Mtx2 mosquitocidal toxin of B. sphaericus SSII-1, the-toxin of Clostridium perfringens, and the cytotoxin of Pseudomonas aeruginosa. The mtx3 gene is highly conserved and widely distributed in both high-and low-toxicity mosquito larvicidal strains of B. sphaericus. More than 380 Bacillus sphaericus strains which are toxic to the larvae of mosquitoes in at least three genera (Culex, Aedes, and Anopheles) have been identified (1, 5). These strains have been subdivided into the low-toxicity strains (50% lethal concentration [LC 50 ], ϳ10 5 cells per ml) and the high-toxicity strains (LC 50 , 10 2 to 10 3 cells per ml) (1, 14). Until recently, only two types of mosquitocidal toxin have been found in B. sphaericus: a binary toxin (comprising 51.4-and 41.9-kDa proteins) produced during sporulation in high-toxicity strains (1) and a 100-kDa toxin synthesized in both the low-and hightoxicity strains (18). To date, few mosquitocidal-toxin genes have been isolated and characterized (13). It is important to isolate and identify new and potent mosquitocidal toxins for use as potential biocontrol agents (14). In this paper, we describe the isolation, characterization, and distribution among B. sphaericus strains of a new type of mosquitocidal-toxin gene. Cloning of the mtx3 gene. Chromosomal DNA was prepared according to a published method (4). ClaI-digested chromosomal DNA fragments were separated in a 1% agarose gel, and fragments above 5 kb were isolated from the gel by using the GeneClean kit (Bio 101, Inc.). The isolated fragments were ligated into AccI-digested, alkaline phosphatase-treated plasmid pUC18 (19) and transformed into competent Escherichia coli DH5␣. Six hundred white recombinant colonies from the library described above were grown overnight at 37ЊC in 1 ml of Luria-Bertani medium containing 100 g of ampicillin per ml. The cells were harvested by centrifugation, washed once with water, and fed to five second-instar larvae of Culex quinquefasciatus. A toxicity test was performed at 30ЊC for 24 h (8). A toxic clone, pS57, with an insert of ϳ7 kb was identified as the parent clone of the new toxin gene. Subclones were produced from pS57 to localize the region within the DNA insert which encodes the protein toxic to C. quinquefasciatus larvae. After XbaI digestion of pS57, a subclone of 3.1 kb, pSX18, was still toxic to larvae. pSX18 was further digested with PstI and KpnI, and the resulting two fragments of 2.0 and 1.