Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5(S34N), or small interfering RNA (siRNA)-mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to alpha-adaptin ear, which displaces the ear-associated mu2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-mu2 levels, and expression of a phosphomimetic mu2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5(S35N) expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P(2)) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating mu2 dephosphorylation and PtdIns(4,5)P(2) levels in CCVs.