Reservoir cells latently infected with HIV-1 pose one of the major obstacles that hamper ultimate eradication of HIV-1 from infected patients. In this report, we showed that direct contact with MOLT-4 T cells induced HIV-1 replication in J(22)-HL-60 latently infected cells without any additional stimulus. Neutralization experiments revealed that pro-inflammatory cytokines, whose production was increased following cell-cell contact, were unlikely to be primarily involved in the induced HIV-1 replication. Cell-cell contact, but not soluble components in the culture supernatant, caused a rapid phosphorylation and degradation of IkappaBalpha, which led to elevated NF-kappaB DNA binding activity in J(22)-HL-60 cells. Furthermore, forced expression of a super-repressor form of IkappaBalpha or pretreatment with ritonavir efficiently blocked the activation of NF-kappaB and HIV-1 replication in J(22)-HL-60 cells co-cultured with MOLT-4 T cells. Moreover, either resting or PHA stimulated primary CD4(+) T cells induced HIV-1 replication in J(22)-HL-60 cells in a similar way with that of MOLT-4 cells. These results indicated that direct contact with CD4(+) T cells induced HIV-1 replication in latently infected cells and provide insight into the molecular mechanism of virus release from myeloid progenitor cells latently infected with HIV-1.