Dendritic spines are the primary sites of excitatory transmission in the mammalian brain. Spines of cerebellar Purkinje Cells (PCs) are plastic, but they differ from forebrain spines in a number of important respects, and the mechanisms of spine plasticity differ between forebrain and cerebellum. Our previous studies indicate that in hippocampal spines cortactin-a protein that stabilizes actin branch points-resides in the spine core, avoiding the spine shell. To see whether the distribution of cortactin differs in PC spines, we examined its subcellular organization using quantitative preembedding immunoelectron microscopy. We found that cortactin was enriched in the spine shell, associated with the non-synaptic membrane, and was also situated within the postsynaptic density (PSD). This previously unrecognized distribution of cortactin within PC spines may underlie structural and functional differences in excitatory spine synapses between forebrain, and cerebellum.