We report here on brain associated autoimmune features in opiate-dependent subjects. This study includes 107 (37 HIV + and 70 HIV -) hospitalized heroin-addicted subjects on a methadone maintenance program, and 45 healthy individuals. Human brain S100 protein, neuron specific enolase (NSE), myelin basic protein (MBF), and old tuberculin (OT) were used as antigens in the study. Serum autoantibodies to brain antigens S100, NSE and MBP were detected by ELISA, whereas delayed hypersensitivity skin reactions were evaluated after intradermal injection of S100, NSE, MBP and OT (control brain-irrelevant antigen). In drug-dependent subjects, 68.2% produced anti-S100, 56.1% anti-NSE and 20.5% anti-MBP autoantibodies, while the incidence of autoantibodies in control healthy individuals was 4.4%, 2.2% and 0%, respectively. Occurrence and amount of anti-S100 and anti-NSE autoantibodies were much higher in HIV + than in HIV - heroin-abusing adults. In drug abusers, the incidence of positive delayed hypersensitivity skin reactions were as follows: 67.2% to S100, 51.4% to NSE, 14.9% to MBP, and 94.3% to OT. In control subjects, the occurrence of hypersensitivity reactions to brain antigens was insignificant. Cutaneous reactions were more frequent in HIV - addicts. The incidence of both autoantibodies and delayed skin responses was positively related to the duration of drug abuse, worsening of HIV infection, and dementia. The high incidence of autoantibodies and delayed hypersensitivity skin reactions to S100 and NSE human brain antigens in heroin-abusers indicates that heroin dependence, as well as HIV infection, are associated with a hyperergy towards brain-related autoimmune phenomena. It has been suggested that the brain-associated autoimmune phenomena in HIV + heroin-addicts represent a hyperimmune phase which precedes immunodeficiency that occurs in the further development of HIV infection.