Acute HIV-1 infection of H9 and C8166 cultures has been shown to be suppressed by certain flavonoids, and evidence for inhibition of HIV-1 protease, integrase, and reverse transcriptase by flavonoids also exists. The present aim was to determine whether flavonoids inhibit HIV-1 activation in models of latent infection. By screening flavonoids from six different classes, three structurally related compounds (chrysin, acacetin, and apigenin) were identified that inhibited HIV expression in TNF-alpha-treated OM-10.1 cultures. The three compounds had favorable potencies against HIV activation in relation to their growth inhibitory effects (therapeutic index 5-10). Chrysin also inhibited HIV expression in response to PMA in OM-10.1 cells, in ACH-2 cells stimulated with either TNF-alpha or PMA, and in 8E5 cultures. Furthermore, return to viral latency in OM-10.1 cells previously exposed to TNF-alpha occurred over a shorter time interval when chrysin was added. The inhibition of HIV activation was not dependent on preincubation with flavonoids relative to TNF, and was characterized by a lack of HIV RNA accumulation by Northern analysis. Gel-shift experiments revealed that NF-kappa B activation after TNF-alpha treatment was not inhibited by these agents, suggesting that some other critical factor(s) needed for viral transcription was being affected. These findings indicate that flavonoids inhibit HIV-1 activation via a novel mechanism, and that these agents are potential candidates for therapeutic strategies aimed at maintaining a cellular state of HIV-1 latency.