Recombinant lentiviral vectors pseudotyped with heterologous HIV-1 envelope glycoproteins allow rapid and accurate measurement of antibody-mediated HIV-1 neutralization. However, the neutralization phenotypes of envelope pseudoviruses have not been directly compared to isogenic replication competent HIV-1. We produced pseudoviruses expressing three different HIV-1 envelope glycoproteins and subcloned the same three env genes into a replication competent NL4-3 molecular clone. For each of the antibodies tested, the neutralization dose–response curves of pseudoviruses and corresponding replication competent viruses were similar. Thus, envelope pseudoviruses can be used to study the anti-HIV-1 neutralizing antibody response. A single passage of replication competent virus derived from 293T cells through peripheral blood mononuclear cells (PBMC) caused a substantial decrease in sensitivity to neutralizing antibodies. This was associated with an increase in average virion envelope glycoprotein content of the PBMC-derived virus. Replication competent HIV-1 and isogenic envelope pseudoviruses have similar neutralization characteristics, but passage into PBMC is associated with decreased sensitivity to neutralization.