Production of a factor causing vacuoles in HEp-2 culture cells by Bacillus cereus No. 55 was examined in a semi-synthetic medium containing ammonium sulfate as a sole nitrogen source (AYPS medium). Although the vacuolation activity in the culture supernatant was low after cultivation of B. cereus in the medium, the activity was increased by supplementation of glucose to the medium. However, the activity did not increase by the supplement of galactose and lactose to the medium. The activity of the factor increased with an increase in the cell concentration of B. cereus when it was cultured with shaking in AYPS medium supplemented with 0.5% glucose. In the medium with 0.1% glucose, the cell concentration reached the highest level at 8 hr, but the activity was the highest at 48 hr. In both media the rate of sporulation was above 65% after a 48hr cultivation with shaking. By contrast, the rate of sporulation was very low (4-15%) without shaking. The activity was first detected at 24 hr and increased until 48 hr to the same level as that in the culture with shaking. For purification of the vacuolation factor, salting out with ammonium sulfate, n -butanol extraction, solubilization with methanol and thin-layer chromatography (TLC) on silicagel 60 were effective. After fractionation by the TLC, the vacuolation activity was detected in five fractions.