Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are essential components in first-line therapy for human immunodeficiency virus (HIV) infection. However, long-term treatment with existing NRTIs can be associated with significant toxic side effects and the emergence of drug-resistant strains. The identification of new NRTIs for the continued management of HIV-infected people therefore is paramount. In this report, we describe the response of a primary isolate of simian immunodeficiency virus (SIV) to 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) both in vitro and in vivo. EFdA was 3 orders of magnitude better than tenofovir (TFV), zidovudine (AZT), and emtricitabine (FTC) in blocking replication of SIV in monkey peripheral blood mononuclear cells (PBMCs) in vitro, and in a preliminary study using two SIV-infected macaques with advanced AIDS, it was highly effective at treating SIV infection and AIDS symptoms in vivo. Both animals had 3- to 4-log decreases in plasma virus burden within 1 week of EFdA therapy (0.4 mg/kg of body weight, delivered subcutaneously twice a day) that eventually became undetectable. Clinical signs of disease (diarrhea, weight loss, and poor activity) also resolved within the first month of treatment. No detectable clinical or pathological signs of drug toxicity were observed within 6 months of continuous therapy. Virus suppression was sustained until drug treatment was discontinued, at which time virus levels rebounded. Although the rebound virus contained the M184V/I mutation in the viral reverse transcriptase, EFdA was fully effective in maintaining suppression of mutant virus throughout the drug treatment period. These results suggest that expanded studies with EFdA are warranted.