Members of the cadherin family of calcium-dependent cell adhesion molecules can bind homophilically across central nervous system (CNS) synapses, but experimental evidence indicates the nature of their contribution to synapse structure and function changes over time. We asked whether changes in function correspond to differences in intrasynaptic distribution. Using quantitative immuno-electronmicroscopy, we determined where cadherins are localized within synapses at key developmental stages in cultured hippocampal neurons and in hippocampus in vivo. At 5–6 days in culture, when most synapses are newly formed, cadherins are regularly and evenly distributed at synaptic clefts throughout the active zone. In contrast, at 14 days, when the majority of synapses are comparatively mature, cadherin labeling concentrates in discrete clusters. Such clusters can occur at any place within or immediately surrounding synaptic clefts. To assess whether this change in distribution is unique to neurons grown in culture, we compared the distribution of cadherins in the CA1 region of hippocampus at postnatal days 2, 3 (P2–3) and in adult. Consistent with our observations in cultured neurons, synapses in P2–3 hippocampus most often exhibit cadherins distributed regularly throughout the cleft, while adult synapses show predominantly discrete concentrations at single sites. The early developmental pattern of cadherin distribution can also be detected at occasional synapses in adult tissue. Such synapses also have morphological features consistent with immature synapses, suggesting that intrasynaptic cadherin distribution is a feature that may distinguish synapse age. J. Comp. Neurol. 495:324–335, 2006. © 2006 Wiley-Liss, Inc.