Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions.