The laminar distribution and cellular levels of expression of mRNAs encoding N-methyl-D-aspartate receptor subunits (NMDAR1, NMDAR2A-D and the alternatively spliced isoforms of NMDAR1) were examined in prefrontal cortex of rat by in situ hybridization using film and emulsion autoradiography. Film autoradiograms demonstrated a distinctive laminar distribution of hybridization signals for each of the probes recognizing NMDAR1, NMDAR2A and NMDAR2B messenger RNA; hybridization with probes for NMDAR2C and NMDAR2D resulted in scattered signals without laminar organization. Grain counting disclosed that neurons in layer V displayed the highest and neurons in layer IV the lowest absolute number of grains for all probes examined. Correction for cell size demonstrated statistically significant differences in cellular labelling density of up to 50% across neurons in different cortical layers. The cellular density profiles across cortical laminae differed between probes. Hybridization with a probe recognizing all isoforms of NMDAR1 resulted in significantly lower densities of cellular labelling in neurons of layer IV than of layers II/III, V and VI. Cellular labelling densities following hybridization with probes recognizing alternatively spliced segments of NMDAR1 were examined. Densities were low in neurons of the upper cortical layers II/III and IV using probes for the messenger RNA encoding the amino terminal insert, NMDAR1(1XX) and the second carboxy terminal deletion, NMDAR1(XX1); hybridization with a probe for the messenger RNA encoding the first carboxy terminal deletion, NMDAR1(X1X), resulted in low cellular signal densities in neurons of layers IV and VIb. NMDAR2A messenger RNA expression was of relatively uniform intensity in neurons of layers II-V but significantly lower in neurons of the inner part of layer VI. NMDAR2B expression was most dense in layer II neurons. These data indicate that neurons in different cortical laminae express distinct N-methyl-D-aspartate receptor subunit messenger RNA phenotypes. In addition, the observed differences in density of N-methyl-D-aspartate receptor subunit messenger RNA expression suggest that cortical laminae differ in the relative contribution of N-methyl-D-aspartate receptors to their excitatory responses.