In utero sensitization to infectious pathogens can establish immunological memory and may influence the immune response to unrelated antigens. Little is known about the influence of intrauterine human immunodeficiency virus (HIV) exposure on the cellular immune response to mycobacterial antigens. Whole-blood culture gamma interferon (IFN-gamma) production in response to mycobacterial antigens was measured at birth and 6 weeks of age to determine the characteristics of the IFN-gamma response in HIV-exposed infants to Mycobacterium bovis BCG and mycobacterial antigens. At birth, we observed an increased immune activation in response to phytohemagglutinin among HIV-exposed, uninfected infants. In a proportion of these infants, we also observed an increased immune activation in response to purified protein derivative, BCG, and early secreted antigen target 6. Increases in the IFN-gamma response to the four antigens between birth and 6 weeks of age, observed in all HIV-unexposed infants, was absent in a substantial proportion of HIV-exposed, uninfected infants. The immunological differences persisted at 6 weeks of age, suggesting a sustained impact of in utero immune priming by HIV. Intrauterine exposure to HIV affects the infants' cellular immune response to mycobacterial antigens, either specifically or as a consequence of nonspecific, broadly reactive immune activation. Further studies will be important to help determine optimal vaccination and disease prevention strategies for this vulnerable population group.