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Comorbidity, aging, and their impact on physical functioning will play an increasingly greater role in HIV medical care as the number of infected adults over 50 years of age grows. The study objective was to investigate the relationship of comorbidity and age with physical functioning in HIV-infected and HIV-negative patients. Eight hundred eighty-nine HIV-infected veterans and 647 HIV-negative veterans from the Veterans Aging Cohort Study conducted between September 2001 and June 2002 were included in the study. Physical functioning was measured by self-reported difficulty with various physical activities. Regression analyses were performed to examine demographic and clinical factors associated with physical functioning. Separate models were used for HIV-infected and HIV negative subjects since these groups differed in demographic makeup. In both patient groups, chronic lung disease, coronary artery disease, hypertension, smoking, and major depression were independently associated with reduced physical functioning in age and race adjusted regression models. Increased age was associated with reduced physical functioning in both HIV-infected and HIV-negative patients. However, when comorbid conditions were entered into the models for both HIV-infected and HIV-negative patients, age coefficients were reduced and were no longer statistically significant. Among the HIV-infected patients, results remained unchanged after controlling for the impact of antiretroviral therapy and HIV disease stage. Our findings demonstrate the important role of general medical comorbidity in physical functioning in both HIV-infected and HIV-negative patients. This suggests the importance of effectively treating comorbid conditions in persons with HIV, in order to reduce the overall impact of disease on physical functioning.