The safety and the immunogenicity of a recombinant hybrid envelope glycoprotein MN/LAI (rgp160) followed by booster injections of a V3 (MN) linear peptide were evaluated in HIV-negative adults at low risk for HIV infection. Volunteers received either rgp160 in alum at 0, 1, and 6 months (group A), rgp160 at 0 and 1 months followed by V3 at 3 and 6 months formulated in alum (group B), or in Freund's incomplete adjuvant (FIA) (group C). Local and systemic reactions were mild to moderate and resolved within the first 72 hr after immunization. No significant biological changes (routine tests and autoantibodies) were observed. One month after the last injection in either group, neutralizing antibodies (NAs) against the HIV-1 MN isolate were detected in 4 of 5 (A), 7 of 10 (B), and 10 of 10 (C) subjects with significantly higher geometric mean titers in the latter group. Four of nine sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI strain or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp160 was detected in 92% of the subjects after the second injection of rgp160 and in 80% of them 12 months after the first injection. A weak and short-lived envelope-specific CD(4+)-mediated cytotoxic lymphocyte activity was detected at certain time points in few subjects. This prime-boost vaccine approach using rgp160 followed by a V3 peptide was safe in humans, and was able to elicit high levels of neutralizing antibodies and a strong and persistent T cell lymphoproliferative response to rgp160 and to V3. However, the neutralization response was restricted to the homologous HIV-1 strain and little env-specific cytotoxic activity was induced.