Mechanism of the production of off-flavor in Brassica vegetables, mainly broccoli stored under anaerobic conditions was studied. Methanethiol and dimethyl disulfide were identified as the volatile sulfur compounds in the headspace of anaerobically stored Brassica vegetables, including broccoli. When the activity of C-S lyase, a key enzyme for the formation of volatile sulfur compounds, was measured, no significant changes were observed in the C-S lyase activity in broccoli kept under anaerobic and aerobic conditions. The most obvious difference between anaerobically and aerobically kept broccoli was the degradation of the cell membranes. These results suggest that under anaerobic conditions volatile sulfur compounds are formed by the degradation of cellular membrane lipids and loss of intracellular compartmentation, allowing the enzyme-substrate reaction to proceed. Furthermore, the factors which affect the production of the volatile sulfur compounds, under anaerobic conditions were also investigated. Methanethiol which was one of the first compounds formed under anaerobic conditions appears to be primarily responsible for the off-flavor. Therefore, the chemical nature of the formation processes of methanethiol was also analyzed.