A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.