The purpose of this study is to examine the association of alcohol abuse and injection drug use (IDU) with the immunologic and virologic responses to highly active antiretroviral treatment (HAART) in urban community health clinics. The medical records of 293 HIV-infected adult patients who visited either of two urban health clinics in New Haven, Connecticut, from June 2003 to December 2004 were retrospectively reviewed. Changes in mean CD4 lymphocyte counts and undetectable viral loads were compared before and after the initiation of HAART for patients categorized into one of four substance abuse groups: history of neither alcohol abuse nor IDU, alcohol abuse only, IDU only, or both. Unadjusted mean improvements in CD4 count for the four groups were 136, 97, 20, and 27, respectively. In a linear regression model adjusted for age, gender, and baseline CD4 count, history of IDU only (P = 0.037) and a combination of alcohol abuse and IDU (P = 0.038) were associated with a lesser increase in CD4 count after HAART compared with those with neither alcohol nor IDU. No significant associations were found between substance abuse history and changes in detectable viral load. Our results show that many patients at urban health clinics have a history of either injection drug use or alcohol abuse, and that injection drug use is negatively associated with the immunologic response to HAART in urban HIV-infected individuals. This study highlights the importance for clinicians of understanding the negative associations of substance abuse with the treatment response of HIV-infected patients at urban health centers.