Regulation of fat cell number by apoptosis is proposed to be part of a normal physiological cycle in adipose growth and development. To investigate this process, cultured rat adipocytes were treated with various concentrations of tumor necrosis factor alpha (TNF alpha) and/or insulin to determine the roles of these factors in adipocyte apoptosis. The cells were analyzed by flow cytometry using a TUNEL assay. TNF alpha increased adipocyte apoptosis in a dose-dependent fashion. TNF alpha-mediated apoptosis was detectable within 6 h of treatment and continued to increase with time. Decreasing media insulin concentration from 8.5 to 0.85 nM resulted in increased adipocyte apoptosis, whereas high doses of insulin protected adipocytes from TNF alpha-induced apoptosis. TNF alpha-activated apoptosis was accompanied by an increase in caspase 3 activity and could be inhibited by a caspase S-specific inhibitor. These data suggest that adipose tissue cell number is regulated, in part, by an apoptotic signaling pathway that involves TNF alpha, insulin, and caspase 3.