Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.