Free folic acid is believed to enter some cells by folate receptor-mediated endocytosis at membrane invaginations termed caveolae. Folate conjugated macromolecules also enter cells by folate receptor-mediated endocytosis, but their site of entry has never been conclusively identified. In this paper, we show that internalization of folate-macromolecule conjugates by receptor-bearing KB cells can be blocked by agents that specifically inhibit caveolae assembly or internalization such as nystatin and phorbol-12-myristate acetate (PMA). To characterize the intracellular conditions to which the macromolecule-folate conjugates are subsequently exposed, we have measured the pH of the major compartments of the folate endocytosis pathway. pH values of individual endosomal compartments in KB cells were determined by dual-excitation laser-scanning confocal microscopy, where the fluorescence ratio of folate-DM-NERF-dextran (pH-sensitive) and Texas Red-dextran (pH-insensitive) was used to calculate pH. These studies revealed that the pH of folate conjugate-containing endosomes commonly varies between 4.7 and 5.8, with the pH in some endosomes as low as 4.3. The most frequent pH value in these compartments was approximately 5.0.