We previously demonstrated that Actinomycin D (ActD) enhanced HIV-1 replication in the MT-2 cell, a human T-cell leukemia virus type-1-infected cell line. The MT-2 cell is known to produce multiple cytokines spontaneously. In this study, we investigated the impact of ActD on the cytokine production from MT-2 cells and HIV-1 replication in a latently infected cell line, U1. MT-2 cells were pulse-treated with 0 or 200 nM of ActD, and culture supernatants were collected 3 days after incubation. Supernatants from untreated cells (Sup0) induced HIV-1 replication by 150-fold in U1 cells. Culture supernatants from ActD-treated cells (Sup200) enhanced HIV-1 replication by 1200-fold. A combination of a sequential chromatographic approach and mass spectrometric analysis identified that the HIV-inducing factors in Sup200 were interleukin (IL)-6 and tumor necrosis factor (TNF)-beta. Quantitative analysis revealed that ActD treatment increased the concentration of IL-6 in Sup200 by 600% compared with that in Sup0 but decreased the amount of TNFbeta in Sup200 by 85%. Northern blot analysis showed that ActD treatment increased IL-6 transcripts; however, no change was seen in TNFbeta transcripts. These results suggest that ActD induces replication of HIV-1 through modulation of cytokine production.