Antibodies generated by candidate HIV-1 vaccines in a phase I clinical trial were assessed for neutralizing activity with a panel of eight well-characterized, genetically diverse clade B primary isolates having an R5 phenotype. The vaccines consisted of one of three different recombinant canarypox vectors expressing membrane-anchored HIV-1(MN)gp120 (ALVAC vCP205, vCP1433, and vCP1452) followed by boosting with a soluble gp160 hybrid consisting of MNgp120 and the majority of gp41 from strain IIIB. Serum samples from a subset of volunteers in each arm of the trial, containing moderate to high titers of neutralizing antibodies to HIV-1 MN, were analyzed. Competition assays with peptides revealed that the majority of neutralizing activity was specific for the MN-V3 loop. Despite MN-specific neutralization titers that sometimes exceeded 1:500, no neutralization of primary isolates was detected and, in some cases, mild infection enhancement was observed. In addition, little or no neutralization of the HIV-1 IIIB heterologous T cell line-adapted strain of virus was detected. These results reinforce the notion that monovalent HIV-1 ENV is a poor immunogen for generating cross-reactive neutralizing antibodies.