Abstract Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.