Stabilization of long-term potentiation (LTP) depends on multiple signaling cascades linked to actin polymerization. We used one of these, involving phosphorylation of the regulatory protein cofilin, as a marker to test whether LTP-related changes occur in hippocampal synapses during unsupervised learning. Well handled rats were allowed to explore a compartmentalized environment for 30 min after an injection of vehicle or the NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). Another group of rats consisted of vehicle-injected, home-cage controls. Vehicle-treated rats that explored the environment had 30% more spines with dense phosphorylated (p) cofilin immunoreactivity in hippocampal field CA1 than did rats in the home-cage group. The increase in pCofilin-positive spines and behavioral evidence for memory of the explored environment were both eliminated by CPP. Coimmunostaining for pCofilin and the postsynaptic density protein 95 (PSD-95) showed that synapses on pCofilin-positive spines were substantially larger than those on neighboring (pCofilin-negative) spines. These results establish that uncommon cellular events associated with LTP, including changes in synapse size, occur in individual spines during learning, and provide a technique for mapping potential engrams.