88 Lipolysis appears to be an important regulator of hepatic glucose production and gluconeogenesis (GNEO). The aim of the present study was to examine the effects of high fat feeding (a diet-induced insulin resistant model) and exercise on this relationship. Thirty-nine male Wistar rats were fed a purified high starch diet (68% of energy from corn starch) for a 2 wk baseline period then randomly assigned to one of four experimental groups; 68% starch/12% corn oil (ST; n=12), 35% starch/45% corn oil (HF; n=13), ST + exercise (ST+EX; n=7), HF + exercise (HF+EX; n=7). Energy intake was matched within sedentary and exercise groups. Exercise animals had free access to running wheels and averaged (rev/d) 2808±471 for ST+EX and 2228±995 for HF+EX. Following an 8 week period, whole-body lipolysis(using 2H5-glycerol), glucose appearance (Glc Ra, using 3-3Hglucose) and alanine GNEO (using U-13Calanine) were estimated in 6-8 hr fasted rats (n=7/group). In a separate group of ST and HF rats (n=5/group), lipolysis (2H5-glycerol) and GNEO using3 H2O (gluconeogenesis from pyruvate) were estimated. Total body weight and body fat were not significantly different between ST and HF, but were significantly lower in exercise (438±12 g and 49±5 g, respectively) vs. sedentary (493±10 g and 77±3 g, respectively) animals. Glc Ra was not significantly different among groups (9.1±1.0 mg/kg/min). However, the ratio of glucose to alanine enrichment was 0.25±0.01 in HF and 0.15±0.01 in ST. Voluntary exercise had no significant effect on alanine GNEO. In addition, the ratio of 3H in carbon 2 vs. carbon 6 of glucose (an estimate of the rate of GNEO from pyruvate relative to total glucose production) was 0.77±0.11 in HF and 0.49±0.11 in ST. Whole-body lipolysis (μmol/kg fat mass/min) was significantly increased in exercised animals: 63.3±6.1 in ST, 79.2±4.2 in HF, 103.9±4.9 in ST+EX, and 123.0±5.6 in HF+EX. Lipolysis explained ≈25% of the variance in GNEO in sedentary animals, and less than 5% of the variance in exercise animals. HF feeding, but not voluntary exercise, increases the contribution of GNEO to Glc Ra. Whole-body lipolysis appears to only explain a minor proportion of the variance of GNEO.