Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with synaptojanin 1 [1,2]. Neurons of synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type synaptojanin 2, but not of wild-type synaptojanin 1 or mutant synaptojanin 2 that is deficient in 5'-phosphatase activity. In addition, electron-microscopic analysis shows that synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that synaptojanin 2 is functionally distinct from synaptojanin 1.