A plasmid-borne gene from Bacillus thuringiensis var. israelensis encoding a 27,340 Mr insecticidal delta-endotoxin has been cloned on a bifunctional multicopy plasmid in a wild-type sporogenic strain and two asporogenic mutants of Bacillus subtilis. The delta-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion. However, in a stage OA mutant, this increase in delta-endotoxin synthesis is not observed. In both the wild-type sporogenic B. subtilis and the asporogenic OJ strain, phase-bright inclusions, resembling the israelensis crystal in appearance, are visible during late stationary phase. The insoluble inclusions from the B. subtilis transformants, consisting solely of the 27,340 Mr polypeptide, were purified by density gradient centrifugation and found to be extremely toxic to Aedes aegypti larvae. After solubilization in alkaline buffer, this polypeptide was also shown to be haemolytic for human erythrocytes and to lyse Aedes albopictus cells with the same LC50 value as native israelensis delta-endotoxin crystals. During stationary phase, novel mRNA species appear in both the wild-type strain and the OJ mutant, but not in the OA mutant, and these appear to be the major gene-specific transcripts. Transcriptional mapping of delta-endotoxin-specific mRNA has shown that the same region of initiation is used at a relatively low level in all three strains during vegetative growth.