In order to explore the relative contributions of the different ionotropic excitatory amino acid receptor subtypes to signalling in primary visual cortex, we have mapped their distributions in area 17 of adult ferret cerebral cortex by quantitative in vitro autoradiography. D,L-alpha-amino-3-hydroxy-5-methoxy-4-isoxazole propionate (AMPA) and kainate receptors, gating fast, Na(+)-permeable channels, were localized with [3H]dizocilpine maleate ([3H]MK-801). All three radioligands bound to single sites, with KDs of 414 nM [3H]AMPA and [3H]kainate, respectively. Slower-acting N-methyl-D-aspartate receptors, which gate the influx of Ca2+ as well as Na+, were localized with ([3H]AMPA), 78 nM ([3H]kainate) and 16 nM ([3H]MK-801), and each receptor subtype displayed a different laminar distribution pattern within area 17. AMPA receptors were concentrated in superficial layers, with intermediate densities in deep layers and lowest levels in layer IV. Kainate receptor levels were high in layers V and VI and low in all other layers. N-methyl-D-aspartate receptors were more homogeneously distributed than AMPA or kainate receptors, but were expressed at highest levels in layers I and IV and lowest levels in layers V and VI. The binding site densities found in the layers containing most receptors were Bmax = 2812 fmol/mg for [3H]AMPA, Bmax = 626 fmol/mg for [3H]MK-801 maleate and Bmax = 278 fmol/mg for [3H]kainate. Thus, while AMPA receptors were predominant and kainate receptors least abundant in all cortical layers, a complementary relative distribution of excitatory amino acid receptors was apparent, with AMPA receptor density highest in superficial layers, kainate receptor density highest in inferior layers and N-methyl-D-aspartate receptor density highest in the middle granular layer, as well as in layer I. The results indicate that although AMPA receptors are principally involved in excitatory signalling in adult ferret primary visual cortex, kainate receptors in layers V and VI and N-methyl-D-aspartate receptors in layers I and IV may have particularly important roles in mediating synaptic transmission.