Aims: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions. Methods and Results: Isolates were identified as B. cereus by 16S rDNA sequencing. External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B. cereus ATCC 10702. Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated. Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10. Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs. Conclusions: Bacillus DL5 was a neutrophile with a growth pH range similar to B. cereus ATCC 10702, but tolerated alkaline pH. This may 6 e a general trait of the B. cereus species rather than a specific phenomenon of isolates from alkaline ecosystems. Significance and Impact of the Study: Other neutrophilic B. cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here. These results may have important implications for dairy manufacturers.