Alterations in plasma membrane function are induced by many cytopathic viruses, including human immunodeficiency virus type 1 (HIV-1). These alterations can result in changes in the intracellular content of ions and other small molecules and can contribute to cytolysis and death of the infected cell. The pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester was used to quantitate intracellular pH (pHi) in HIV-1-infected T cells. Infection of cells from the CD4+ T-lymphoblastoid line HUT-78 (RH9 subclone) with HIV-1 strain LAI resulted in a significant decrease of pHi, from approximately 7.2 in mock-infected cells to below 6.7 by day 4 after infection, when cells were undergoing acute cytopathic effects. The pHi in persistently infected cells that survived the acute cytopathic effects of HIV-1 was approximately 6.8 to 7.0. Studies with amiloride, an inhibitor of the Na+/H+ exchange system, suggest that HIV-1-induced intracellular acidification in lymphocytes is due, in part, to dysfunction of this plasma membrane ion transport system. The alterations in pHi may mediate certain cytopathic effects of HIV-1, thereby contributing to depletion of CD4+ T lymphocytes in patients with AIDS.