Reversal of proviral latency is being pursued as a curative strategy for HIV-1 infection. Recent clinical studies of in vivo administration of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) show increases in unspliced cellular HIV-1 RNA levels in resting CD4⁺ T cells. A critical unknown, however, is the proportion of latent proviruses that can be transcriptionally reactivated by SAHA or T-cell activation. In this study, we quantified the fraction of HIV-1 proviruses in resting CD4⁺ T cells from patients on suppressive antiretroviral therapy that were reactivated ex vivo with SAHA or antibodies to CD3/CD28. At concentrations of SAHA achieved clinically, only 0.079% of proviruses in resting CD4⁺ T cells were reactivated to produce virions, compared with 1.5% of proviruses in cells treated with anti-CD3/CD28 antibodies after correcting for spontaneous virion production in the medium control. A significant positive correlation (ρ = 0.67, P < 0.001) was found between levels of virions in the supernatant and unspliced cellular HIV-1 RNA following anti-CD3/CD28 treatment, but not following SAHA treatment (ρ = 0.21, P = 0.99). These results reveal that the majority of HIV-1 proviruses are not reactivated by current therapeutic approaches and that more effective means of reversing proviral latency will likely be required to deplete HIV-1 reservoirs.