ABSTRACT Persistent HIV replication within active viral reservoirs may be caused by inadequate antiretroviral penetration. Here, we used mass spectrometry imaging with infrared matrix-assisted laser desorption–electrospray ionization to quantify the distribution of efavirenz within tissues from a macaque dosed orally to a steady state. Intratissue efavirenz distribution was heterogeneous, with the drug concentrating in the lamina propria of the colon, the primary follicles of lymph nodes, and the brain gray matter. These are the first imaging data of an antiretroviral drug in active viral reservoirs