The morphological relationship between a synapse and dendritic spine is an important determinant of synaptic function, and has been studied extensively with the electron microscope. This has revealed that the universal idea of one synapse per spine is no longer tenable; dendritic spines may on occasions form synapses with two or more axonal boutons. To elucidate the detailed structure and relationship of spines and their synaptic contacts in the molecular layers of the dentate gyrus of the adult rat, a study using 3-dimensional (3D) reconstructions was made from serial ultrathin sections (up to 150 sections per series), of both pre- and postsynaptic segments of axons and dendrites. We found that synapses from all unmyelinated axons examined were en passant and although most contacts between synapse and spine are one-to-one, all reconstructions examined contained both multisynaptic axonal segments, and multisynaptic spines. In the dentate gyrus, 3.48% mushroom spines were multisynaptic, synapsing with more than two axons, and 0.46% of the total population of synapses were multisynaptic. Although these values appear small, they actually comprise millions of contacts per cubic millimeter of tissue. Axonal varicosities can contact more than three dendritic spines originating from different dendritic branches while spines may branch with up to three heads, and spine heads may receive two or more synaptic contacts. Our data confirm the complexity of relationships between spines and synapses and show that a one-one relationship between synapse and spine cannot be assumed automatically.