Abstract The toxicity of Bacillus thuringiensis subsp. israelensiswas measured in vitro by several assay methods. The chromium-release assay was more advantageous requiring less time and has less error and more accuracy than the trypan blue exclusion method, although no difference in cytotoxicity was observed between the two methods. Although less sensitive, the hemoglobin release assay was fast, simple, and an inexpensive method to determine hemolytic activity. The thymidine-release assay was significantly less sensitive showing no cytotoxicity at doses at which the assays above were sensitive. These assays show that the cytotoxic activity of the solubilized toxins is probably due to pore formation in cell membranes. B. thuringiensisisraelensis parasporal bodies dissolved in 50 m m Na 2CO 3 · HCl ( pH 10.5) showed higher cytotoxicity than parasporal bodies solubilized in 50 m m NaOH/10 m m EDTA ( pH 11.7), and both of these two solubilization procedures gave greater cytotoxicity than parasporal bodies dissolved in 0.1 m glycine · NaOH ( pH 10)/0.01 m EDTA/1 m m PMSF/with 0.1 m 2-mercaptoethanol or 0.1 m dithiothreitol. A predominant protein band of 25 kDa was observed with Na 2CO 3 · HCl solubilization, whereas no distinctive protein band of a similar molecular weight was evident with glycine/EDTA/DTT solubilization. The solubilized toxin was toxic to cells derived from Aedes albopictus, Culex tarsalis, Mardin-Darby canine kidney fibroblasts, and Spodoptera frugiperda. The in vitro toxicity for the latter two cell types occurs at significantly higher doses than for Ae. albopictus cells. The in vitro Ae. albopictus assay gave a sharp dose-response curve with both the crude toxin and purified 25-kDa toxin, and the cytotoxicity was dependent on time of cell exposure to the toxin. However, the Ae. albopictus cells showed a lower sensitivity to purified 25-kDa toxin than crude toxins.