Dynamin GTPases (Dyn1 and Dyn2) are indispensable proteins of the core clathrin-mediated endocytosis (CME) machinery. Best known for their role in fission at the late stages of CME, many studies have suggested that dynamin also plays a regulatory role during the early stages of CME; however, detailed studies regarding isoform-specific early regulatory functions of the dynamins are lacking. With a recent understanding of the regulation of Dyn1 in nonneuronal cells and improved algorithms for highly sensitive and quantitative analysis of clathrin-coated pit (CCP) dynamics, we have evaluated the differential functions of dynamin isoforms in CME using domain swap chimeras. We report that Dyn1 and Dyn2 play nonredundant, early regulatory roles during CME in nonneuronal cells. The proline/arginine-rich domain of Dyn2 is important for its targeting to nascent and growing CCPs, whereas the membrane-binding and curvature-generating pleckstrin homology domain of Dyn1 plays an important role in stabilizing nascent CCPs. We confirm the enhanced ability of dephosphorylated Dyn1 to support CME, even at substoichiometric levels compared with Dyn2. Domain swap chimeras also revealed previously unknown functional differences in the GTPase and stalk domains. Our study significantly extends the current understanding of the regulatory roles played by dynamin isoforms during early stages of CME.