The purpose of this study was to evaluate the relationship of respiratory quotient (RQ), a surrogate marker of substrate oxidation, as well as body composition and dietary intake to resting energy expenditure (REE) among HIV-infected patients in the current era of highly active antiretroviral therapy and among non–HIV-infected control subjects. Resting energy expenditure is increased in HIV-infected patients; but little is known regarding the potential contribution of altered substrate metabolism, body composition, and dietary intake to increased energy expenditure in this population. Respiratory quotient, REE, body composition, and dietary intake parameters were assessed in 283 HIV-infected patients and 146 community-derived HIV-negative controls who were evaluated for metabolic studies between 1998 and 2005. Respiratory quotient was lower (0.83 ± 0.00 vs 0.85 ± 0.01, P = .005), whereas REE adjusted for fat-free mass (FFM) was higher (31.8 ± 0.3 vs 29.8 ± 0.3 kcal/[d kg], P ≤ .0001), in HIV-infected compared with control subjects. In multivariate modeling among HIV-infected patients, including age, sex, and parameters of immune function, FFM (β = 24.811334, P < .0001), visceral adiposity (β = .7182746, P = .008), and total body fat (β = 8.0506839, P = .041) were positively associated with REE, whereas RQ was negatively associated with REE (β = −528.4808, P = .024). Overall r² was equal to 0.705 and P was less than .0001 for the model. In control subjects, by contrast, only visceral adiposity (β = 1.0612073, P = .004), total body fat (β = 15.805547, P = .010), and FFM (β = 22.613005, P < .0001) were significant predictors of REE; and there was no relationship with RQ. Overall r² was equal to 0.825 and P was less than .0001 for the model. These data suggest that alterations in substrate metabolism may contribute to increased REE in HIV-infected patients compared with control subjects.