To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-α) under control of the mycobacterial heat shock protein (hsp)60 promoter and the α antigen signal sequence. Expression of rhIFN-α was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN-α BCG was substantially more active in inducing the production of IFN-γ and IFN-inducible protein 10 (IP-10) from human peripheral blood mononuclear cells, while IL-10 production was correspondingly decreased. These effects were reversible upon antibody neutralization of rhIFN-α. Among 10 patients tested, rhIFN-α BCG enhanced IFN-γ production in all patients ranging from 1·4- to 23·7-fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG. Correspondingly, rhIFN-α BCG decreased IL-10 production in all patients by 1·2–4·8-fold. The onset of IFN-γ production induced by rhIFN-α BCG was also more rapid, occurring within 4 h after stimulation versus > 24 h with wild-type BCG. The observation that the maximum IFN-γ induction depends on the simultaneous presence of both IFN-α and BCG highlights the advantages of rhIFN-α BCG. Taken together, these immunostimulatory properties of rhIFN-α BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.