The distribution of the glutamate receptor subunit GluR1 was investigated in cultured hippocampal neurons by confocal microscopy, using polyclonal antibodies directed against either the N- or C-terminal region. On living neurons, GluR1 immunofluorescence was detected with the N-terminal antibody only. GluR1 was localized in a highly punctate manner on the surface of neuronal soma and throughout the dendritic tree. Many GluR1 puncta co-localized with the synaptic marker synaptophysin, although extrasynaptic GluR1 puncta were also observed. A comparison of GluR1 subunit distribution of living neurons labelled with N-terminal antibody with that obtained after the cells had been fixed, permeabilized and subsequently reacted with C-terminal or additional N-terminal antibody showed a number of differences. In permeabilized cells additional, diffuse labelling was observed which was very pronounced in the soma and extended into the proximal dendrites. Furthermore, some spines showed little or no labelling of their membrane surface, but labelled strongly after the cells had been fixed and permeabilized. Such spines may be the postsynaptic components of silent or suboptimal synapses.