Quantitative live-cell TIR-FM has revealed a striking heterogeneity in the dynamic behavior of clathrin-coated pits (CCPs). Many nascent CCPs abort without maturing to form productive clathrin-coated vesicles (CCVs). We have shown that CCP maturation is governed by cargo, adaptors and other endocytic adaptor proteins (EAPs), leading us to propose the existence of an ‘endocytic checkpoint’ that monitors cargo recruitment, curvature generation, and coat assembly and controls the turnover of nascent CCPs (Loerke, Metten et al., PLoS Biol., 2009). Using siRNA knockdown and reconstitution, together with quantitative single and dual-channel total internal reflection fluorescence microscopy and mathematical modeling (in collaboration with Gaudenz Danuser), we are defining the molecular basis for CCP heterogeneity and for regulation of the endocytic checkpoint. This work is funded by NIH grants GM73165 and MH61345.