The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.