Vasoactive intestinal polypeptide (VIP) has been shown to be present in a morphologically heterogeneous subpopulation of interneurons in the dentate gyrus, but the relationship between their input and output characteristics and neurochemical features has not been established. Three types of VIP-immunoreactive cells have been identified on the basis of these criteria: (i) cells forming a dense axonal plexus in the hilus have always coexisted with the calcium binding protein calretinin (CR), but never with the neuropeptide cholecystokinin (CCK). The postsynaptic targets of these VIP-positive cells were neurons visualized by immunostaining for substance P receptor, which is known to label different hilar non-principal cells. (ii) VIP-immunoreactive basket cells, innervating predominantly the somata and proximal dendrites of granule cells, were found in the striatum moleculare and stratum granulosum. They contained CCK, but not CR. (iii) Cells projecting to the stratum moleculare were found to have dendrites and axons restricted to this layer. In 75% of these cells VIP coexisted with CR but not with CCK, and they established multiple contacts largely with non-principal cells. GABA was shown to be present but the calcium-binding proteins calbindin D28K and parvalbumin were absent in all three types of VIP-containing interneuron. On the basis of these observations we conclude that three different types of VIP-positive neuron are present in this area, and are likely to subserve different inhibitory functions, cells with a hilar projection as well as those projecting to the stratum moleculare may synchronize the activity of hilar and other interneurons, or disinhibit granule cells by specific interneuron-to-interneuron connections. In contrast, basket cells control the activity of granule cells directly, via perisomatic inhibition.