Benzylated derivatives of peptides corresponding to residues 81 through 92 of the CD4 molecule [CD4-(81-92)] inhibit human immunodeficiency virus 1 (HIV-1)-induced cell fusion and infection in vitro. If such peptides are to be considered as candidates in the therapy of HIV infection, it is crucial to know if the anti-HIV efficacy of CD4-based peptides is limited to blockade of infection and virus-induced cell fusion or if other stages of the viral life cycle are affected by these compounds. Accordingly, an in vitro quantitative microassay for acute HIV infection was divided into two kinetic phases corresponding to the two general stages of the viral life cycle: (i) viral infection and (ii) transmission of virus and viral protein products through cell contact or release of free virions. CEM-SS cell cultures were treated with peptide during either the infection or the transmission phase of the assay. When peptides were present during the infection phase, inhibition of syncytium formation correlated with decreased expression of viral core protein p24 and lack of infectious cell centers when cells exposed to virus were washed and replated onto fresh uninfected indicator cells. These data are consistent with complete inhibition of viral infection when peptide is present only during initial exposure to virus. Unexpectedly, parallel inhibition of syncytium formation, decreased p24 levels, and inhibition of infectious cell center formation were also seen even when peptides were added as late as 48 hr after inoculation, during the transmission period of the assay. Since viral binding and penetration are completed well before 48 hr in this assay system, CD4-(81-92) peptide derivatives appear to exert a virostatic effect on cultures already infected with HIV-1, decreasing p24 production, cytopathicity, and cell-mediated infectivity.