Menaquinone-7 (MK-7) is a highly bioactive homologue of vitamin K. We obtained a diphenylamine-resistant mutant strain D200-41 from Bacillus subtilis strain MH-1 which was isolated from fermented soybeans, natto. The mutant strain exhibited decreased production of MK-6. Using strain D200-41, efficient production of MK-7 was achieved. We found that, compared with an agitated and aerated culture, production of MK-7 was increased by static culture. The sporulation of the cells progressed more slowly in a static culture than in an agitated culture. The maximum concentration of MK reached about 60 mg/l in a medium containing 10% soybean extract, 5% glycerol, 0.5% yeast extract and 0.05% K2HPO4 (pH 7.3) when D200-41 cells as well as MH-1 cells were statically cultured at 45 degrees C for 5 d after being cultured with shaking at 37 degrees C for 1 d.