Background: The protease inhibitor (PI) ritonavir (RTV) has been associated with elevated resting lipolytic rate, hyperlipidemia and insulin resistance/glucose intolerance. The purpose of this study was to examine relationships between lipolysis and fatty acid oxidation during rest, moderate exercise and recovery and measures of insulin sensitivity/glucose tolerance and fat redistribution in HIV+ subjects taking RTV (n=12), taking HAART but no PI (n=10) and HIV-seronegative controls (n=10). Methods: Stable isotope tracers [1-¹³C]palmitate and [1,1,2,3,3-²H₅]glycerol were continuously infused with blood and breath collection during 1-hr rest, 70min submaximal exercise (50% VO_2peak) and 1-hr recovery. Body composition was evaluated using DEXA, MRI and MRS, and 2-hr oral glucose tolerance tests with insulin monitoring were used to evaluate glucose tolerance and insulin resistance. Results: Lipolytic and FA oxidation rates were similar during rest and recovery in all groups, however were lower during moderate exercise in both HIV-infected groups (Glycerol Ra: HIV+RTV: 5.1 ± 1.2 vs. HIV+noPI: 5.9 ± 2.8 vs. Cntl: 7.4 ± 2.2 µmol/kgFFM/min, Palmitate oxidation: HIV+RTV: 1.6 ± 0.8 vs. HIV+noPI: 1.6 ± 0.8 vs. Cntl: 2.5 ± 1.7 µmol/kgFFM/min, p<0.01). Fasting and orally challenged glucose and insulin values were similar among groups. Conclusions: Lipolytic and FA oxidation rates were blunted during moderate exercise in HIV+ subjects taking HAART. Lower FA oxidation during exercise was primarily due to impaired plasma FA oxidation with a minor contribution from lower non-plasma FA oxidation. Regional differences in adipose tissue lipolysis during rest and moderate exercise may be important in HIV, and warrant further study.