Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry-liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.