Mitral and tufted cells of the olfactory bulb receive strong γ-aminobutyric acid (GABA)-ergic input and express GABAA receptors containing the α1 or α3 subunit. The distribution of these subunits was investigated in rats via multiple immunofluorescence and confocal microscopy, by using gephyrin as a marker of GABAergic synapses. A prominent immunoreactivity was detected throughout the external plexiform layer (EPL) and glomerular layer (GL). However, although staining for the α1 subunit was uniform throughout the EPL, that of the α3 subunit was most intense in the outer one-third of this layer. All mitral cells were positive for the α1 subunit. In contrast, the α3 subunit was restricted to a subpopulation of mitral cells, many of which also expressed calretinin. Likewise, external tufted cells could be subdivided into distinct groups, either singly labeled for the α1 or α3 subunit or doubly labeled. At the subcellular level, staining for the α1 and α3 subunits was punctate, forming clusters partially colocalized with gephyrin. However, many α1- and α3-positive clusters lacked gephyrin, suggesting the existence of either nonsynaptic GABAA receptor clusters or synaptic receptors not associated with gephyrin. Quantitative analysis of colocalization among the three markers in the inner EPL, outer EPL, and GL revealed considerable heterogeneity, suggestive of a differential organization of GABAA receptor subtypes in the apical and basal dendrites of mitral and tufted cells. Together these results reveal a complex subunit organization of GABAA receptors in the olfactory bulb and suggest that mitral and tufted cells participate in different synaptic circuits controlled by distinct GABAA receptor subtypes. J. Comp. Neurol. 484:121–131, 2005. © 2005 Wiley-Liss, Inc.