In our ecological and serological survey of Bacillus thuringiensis in the Philippines (L. E. Padua, B. P. Gabriel, K. Aizawa, and M. Ohba, Philipp. Entomol. 5, 1% 208, 1982), an isolate designated PG-14 (subsp. morrisoni, serotype H 8a:8b) exhibited rapid and high toxicity to mosquito larvae but showed no toxicity to the larvae of the silkworm, Bombyx mori, or to adults of a daphnid (L. E. Padua, M. Ohba, and K. Aizawa, J. Znvertebr. Pathol. 44, 12-17, 1984). The crystal produced by the isolate PG-14 is morphologically similar to that of B. thuringiensis subsp. israelensis (L. E. Padua et al., 1984,loc. cit.; J. E. Ibarra and B. A. Federici, FEMS Microbial. Lett. 34, 79-84, 1986), and toxicity to mosquito larvae is about equal. It is, therefore, of particular interest to investigate the relationship between crystals produced by these mosquito-specific B. thuringiensis strains. In the present paper, we report the results of comparative crystal serology of these two strains. Type strains of B. thuringiensis subsp. kurstaki (serotype H 3a:3b), subsp. aizawai (serotype H 7), subsp. morrisoni (serotype H 8a:8b), and subsp. israelensis (serotype H 14) were obtained from Dr. H. de Baijac, Institut Pasteur, Paris, France. The isolates AO-50 (subsp. aizawai, serotype H 7) was obtained from the silkworm litter and have been maintained in the Institute of Biological Control, Kyushu University. The isolate PG-14 was obtained from a soil sample in the Philippines (L. E. Padua et al., 1982, lot. cit.). Bacteria were grown in a Sakaguchi flask containing 150 ml of nutrient broth, pH 7.4, with continuous shaking at 25°C for 5 days. The spore-crystal mixture was harvested by centrifugation at 3000 rpm for 20 min at 4”C, washed three times with saline by centrifugation, and treated with ultrasonication for 10 min. Crystals were separated from the spores and cellular debris by 40-50% (w/v) NaBr density gradient centrifugation (G. J. Ang and K. W. Nickerson, Appl. Environ. Microbial. 36,625-626, 1978) using a Spinco Sw25-1 rotor at 30,OOOg for 30 min at 4°C. The purity of crystals was more than 99% when examined under a phase-contrast microscope. The purified crystals were lyophilized and stored at -20°C until use. One milligram (dry weight) of the purified crystals was suspended in the mixture of 750 pl of 0.2 M glycineNaOH buffer, pH 10.2, and 150 pJ of 0.1 M dithiothreitol (DTT). After incubation at 25°C for 1 hr with constant shaking, undissolved material was removed by centrifugation at 10,000 t-pm for 10 min at 4°C. Rabbits were immunized with two subcutaneous injections of solubilized crystals emulsified with an equal volume of Freund’s complete adjuvant (Difco) at weekly intervals. One week later, these were followed by intravenous injections at 4-day intervals with increasing doses of 1, 2, and 3 mg of crystals. A total of 9 mg of crystals was used for each rabbit. Rabbits were bled 1 week after the last intravenous injection. Antisera were heated at 56°C for 30 min and stored at 20°C. Antibody titers of the antisera against crystals of the six B. thuringiensis strains were 1:32-164 when estimated by agarose double immunodiffusion. Antiserum to the purified mosquito-toxic 67-kDa protein (K. H. Kim, M. Ohba, and K. Aizawa, J. Znvertebr. Pathol. 44, 214-