To elucidate the molecular basis of coated pit function, we are developing a method for reconstituting the endocytic cycle in a cell-free system. Using purified membranes attached to a solid substratum, we have successfully reconstituted coated pit assembly. We now show that the endogenous coated pits on these membranes spontaneously round up and leave the membrane at 37°C. As measured by radioimmunoassay, clathrin loss is both pH- and temperature-dependent. The loss is also inhibited by pretreating the membranes with apyrase at 37°C, which suggests that nucleotide triphosphates are required.