Disulfiram (DSF) was well tolerated and activated viral transcription (cell-associated unspliced (CA-US) and plasma human immunodeficiency virus (HIV) RNA) in a phase II dose-escalation trial in HIV+ antiretroviral therapy (ART)-suppressed participants. Here, we investigated whether exposure to DSF and its metabolites predicted these changes in HIV transcription. Participants were administered 500 (N = 10), 1,000 (N = 10), or 2,000 (N = 10) mg of DSF for 3 consecutive days. DSF and four metabolites were measured by ultraperformance liquid chromatography-tandem mass spectrometry. Changes in CA-US and plasma HIV RNA were quantified by polymerase chain reaction (PCR) and analyzed in NONMEM. A seven-compartment pharmacokinetic (PK) model demonstrated nonlinear elimination kinetics. The fitted median area under the curve values for 72 hours (AUC 0–72 ) were 3,816, 8,386, and 22,331 mg*hour/L, respectively. Higher exposure predicted greater increases in CA-US (maximum effect (E max ) = 78%, AUC 50 = 1,600 μg*hour/L, P = 0.013) but not plasma HIV RNA. These results provide support for further development of DSF as an important drug for future HIV cure strategies.