Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.