Cachexia in cancer is characterized by progressive emaciation involving depletion of host adipose tissue stores, the molecular mechanism of which remains largely unknown. In this study, we have attempted to clarify the biologic characteristics of lipid-depleting factor in a mouse cachexia model. Utilizing differentiated 3T3-L1 adipocytes, we established an assay method quantifying the lipid-depleting activity in plasma derived from colon-26-inoculated mice and then analyzed the associated molecular mechanism. Injection (s.c.) of a mouse colon adenocarcinoma cell line, colon-26 clone 20, induced cachexia, as evidenced by progressive weight loss. Addition of clone 20-derived cachexigenic, but not clone 5-derived noncachexigenic, plasma to the culture medium of differentiated 3T3-L1 adipocytes reduced the TG content in cultured cells. The ability of the introduced plasma to induce TG loss in 3T3-L1 cells paralleled the body weight changes of tumor-inoculated host mice. Clone 20 plasma, but not clone 5 plasma or recombinant IL-6, elicited lipolytic activity, which induced glycerol release from 3T3-L1 cells. Addition of clone 20 plasma to cultured 3T3-L1 adipocytes reduced TG synthesis from [(14)C]-glucose compared to clone 5 plasma, indicating that the lipid-depleting activity resulting from addition of clone 20 plasma depended not only on induction of lipolysis but also on inhibition of lipogenesis. Addition of clone 20 plasma to cultured 3T3-L1 adipocytes reduced the quantity of mature SREBP-1 in the nucleus of 3T3-L1 cells without affecting PPAR-gamma and C/EBP-alpha. Although TNF-alpha induced apoptosis in 3T3-L1 cells, clone 20 plasma did not. These results suggest that the lipid-depleting factor in clone 20 plasma is different from either IL-6 or TNF-alpha, and that this factor interfered with not only lipolysis but also lipogenesis through SREBP-1 of 3T3-L1 adipocytes.