Organotypic slice cultures of rat hippocampus are widely used as experimental preparations for the study of synaptic plasticity, but their degree of correspondence with intact brain is not fully known. Here, using postembedding immunogold labelling, we describe the ultrastructural distribution of AMPA-type glutamate receptors (GluR1-4) in CA3 stratum radiatum of organotypic hippocampal slice cultures at 10 days to 11 weeks in vitro and compare the labelling with intact brain of corresponding age. In both types of preparation, the 11-week-old samples contained the highest proportion of AMPA receptor-like immunoreactive synapses. The incidence of labelled synapses, however, was higher in vivo (49%) than in vitro (24%). The intensity of labelling (number of gold particles per labelled synapse) also increased with age and was also higher in vivo than in vitro. In both organotypic cultures and intact brain, labelling was frequently found at presynaptic sites, often attached to vesicular structures. The specificity of these findings was supported both by light microscopic immunolabelling of GluR2/3 subunits and by electron microscopic double labelling of different epitopes of the GluR2 subunit. The vesicular localization of AMPA receptors was supported by Western blot analysis of subcellular fractions. Morphological evidence of presynaptic excitatory innervation of glutamatergic neurons supports a functional role for presynaptically located AMPA receptors. Our results therefore suggest that AMPA receptors occur in both pre- and postsynaptic profiles and that the distribution of AMPA receptors in cultured brain slices is fundamentally similar to intact brain, but that synaptic maturation may be retarded in vitro.