High-pungency red pepper and capsaicin modulate circulating hormone levels and induce lipolysis in adipose tissue in vivo. This study was designed to investigate the lipolytic activity of adipocytes by high-pungency red pepper extract in vitro. High-pungency red pepper (var. Chungyang) powder showed 126.1 mg% of capsaicinoid which was 3 x higher than low-pungency red pepper powder (var. Daemyung). To study the effects of high-pungency red pepper extract on lipolytic activity, preadipocytes were separated from the epidermal fat of 14 day-old rats, induced to differentiate into adipocytes and were treated with red pepper extracts. The amount of glycerol released from adipocytes into the culture medium was analysed to measure lipolytic activity. Glycerol release from adipocytes was increased in a dose-dependent manner with high-pungency red pepper extract treatment. However, there was no significant change in the glycerol release when adipocytes were treated with low-pungency red pepper extract. To investigate whether lipolysis by high-pungency red pepper extract is caused by capsaicin, glycerol release was detected after the treatment of adipocytes with capsaicin. Glycerol release was significantly increased by capsaicin. These results suggest that high-pungency red pepper extract might have a direct lipolytic activity in adipocytes that is mediated by capsaicin.