To determine whether menopausal status affects systemic lipolysis, we measured plasma glycerol concentrations following an overnight fast and during euglycemic hyperinsulinemic conditions (40 mU [middot] m[minus ]2 [middot] min[minus ]1) in 43 middle-aged, premenopausal women (mean [plusmn] SE; 47 [plusmn] 0.4 years) and 26 early, postmenopausal (51 [plusmn] 0.8 years) women. In addition, body composition was measured by dual-energy x-ray absorptiometry and abdominal fat distribution by computed tomography (CT). Postmenopausal women had greater amounts of whole body (fat mass, 22.8 [plusmn] 1.4 v 17.4 [plusmn] 1.2 kg; percent fat, 34.7 [plusmn] 1.2 v 29.1 [plusmn] 1.4; both P [lt ] .01) and intra-abdominal fat (89.0 [plusmn] 6.5 v 55.9 [plusmn] 4.4 cm2; P [lt ] .01) compared with premenopausal women. Despite greater adiposity, plasma glycerol concentrations were similar between pre- and postmenopausal women following an overnight fast (142.7 [plusmn] 9.7 v 136.1 [plusmn] 6.4 [mu ]mol/L) and at 30 minutes (112.7 [plusmn] 5.5 v 108.4 [plusmn] 4.5 [mu ]mol/L ) and 120 minutes (92.7 [plusmn] 4.5 v 97.5 [plusmn] 5.9 [mu ]mol/L ) into the euglycemic hyperinsulinemic clamp. Plasma glycerol levels remained similar after statistical adjustment for fat mass, percent fat, and intra-abdominal fat. Moreover, no differences in plasma glycerol were observed in pre- and postmenopausal women matched ([plusmn] 5%) for fat mass (n = 22/group) or intra-abdominal fat (n = 15/group). In premenopausal women, plasma glycerol levels at 30 and 120 minutes of hyperinsulinemia were positively related to adiposity measures (range, r = .314 to r = .493; P [lt ] .05 to P [lt ] .01), although no relationships were found in postmenopausal women. Our results suggest no effect of menopausal status on plasma glycerol levels under postabsorptive or hyperinsulinemic conditions.