Controversy exists as to whether effects of HIV infection can be detected in the cognitive profiles of substance users, with methodological differences in degree of control for confounding factors a major contributor to empirical discrepancies. To address this shortcoming, we conducted a small but well-controlled study aimed at isolating HIV neurocognitive (NC) effects in a group of chronic substance users. Thirty HIV-negative substance users were individually matched to 30 HIV-positive substance users on relevant medical and demographic factors, including reading level and methadone therapy status. Results revealed that reading level, methadone maintenance therapy, and positive urine toxicology each exerted significant influence on NC function, and that HIV status was a significant predictor of learning and speeded processing after these control factors were considered. The HIV-positive group also displayed significantly more neurologically assessed motor impairment (p < .05), which was specifically related to impaired cognition in this group and independent of degree of immunocompromise. These data demonstrate the need for increased attention to clinical/demographic characteristics of groups under study. They also show that with applied methodological rigor, the deleterious effects of HIV on cognition can be parsed from substance use, even in small samples with chronic and active use histories.