The purpose of the current study was to examine the enzymatic profile [phosphofructokinase (PFK), β-hydroxyacyl-CoA dehydrogenase (HADH), and citrate synthase (CS)] in gastrocnemius muscle, heart, and liver in rats allowed ad libitum access to a high-fat diet (HFD, 45% of kcal from corn oil). Male Wistar rats were fed a low-fat diet (LFD, 12% of kcal from corn oil) for a 2-wk baseline period after which some continued on the LFD and others were placed on the HFD. After 1 wk on the HFD, rats were categorized as obesity-resistant (OR), -intermediate (OI), or -prone (OP) on the basis of body weight gain (OR, lower tertile; OI, middle tertile; OP, upper tertile). At 1, 2, and 5 wk, rats from each group were killed (n = 9-14 from each group/time point) after a 24-h fast. At the end of the 5-wk dietary period, weight gain was 114.8 ± 4.3 in LFD, 125.2 ± 3.7 in OR, 147.1 ± 4.1 in OI, and 173.7 ± 3.5 g in OP rats (OP > OI > OR, LFD; P < 0.001). Energy intake was highly correlated with weight gain on the HFD at each time point (r ≤ 0.72, P < 0.001). After 1 wk on the HFD, significant correlations between the ratio of PFK/HADH (an indication of the relative capacity for glycolysis vs. β-oxidation, r = 0.4, P = 0.03) and HADH/CS (an indication of the capacity for β-oxidation relative to total oxidative capacity, r = 0.56, P = 0.001) in the gastrocnemius muscle and weight gain were observed. At week 2, significant correlations between these ratios and weight gain were observed in the gastrocnemius, liver, and heart. In contrast, these ratios were not significantly correlated with weight gain at 5 wk. These results suggest that rats most susceptible to weight gain on a HFD are characterized by a continuous increase in energy intake (explaining ~50% of the variance in weight gain) and an early tissue enzymatic profile that favors carbohydrate over fat use.