Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CAI pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.