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This study evaluated the effect of continued heroin use during methadone treatment on serum neopterin levels in 40 human immunodeficiency virus (HIV) type 1 seropositive (HIV + ) and 70 seronegative (HIV - ) intravenous drug users (IVDUs). Persistent drug use, determined by urinary evidence of opiates, was more common in HIV+ than in HIV- IVDUs (p = 0.01). Serum neopterin concentration, an indicator of increased probability of progression to AIDS, was elevated in HIV+ IVDUs, p < 0.0001 (mean 16.0 nmol/L) compared to that of HIV - IVDUs (mean 10.1 nmol/L) and in persistent IV drug users compared with abstainers, p < 0.0001. The effect of drug use and HIV status on serum neopterin was not explained by differences in methadone treatment, age, sex, or total years of i.v. drug use. Neopterin decreased in 25 IVDUs, regardless of HIV serostatus, treated with methadone for an average of 1.5 years.