Physiological and immunocytochemical evidence indicates a layer-dependent organization of inhibitory circuits in the neocortex. To investigate the contribution of GABAergic inhibition to frequency tuning in the different cortical layers, we recorded single and multiple units in near-radial penetrations before and during iontophoretic application of the GABA(A)-receptor antagonist bicuculline in the auditory cortex of the lightly anesthetized gerbil. Bicuculline generally increased the spontaneous rate and enhanced and prolonged onset activity. Application of bicuculline often resulted in a shift of best frequency and a decrease of threshold (5.5 dB). A broadening of the frequency tuning evident by lower Q40dB values was observed in 63% of the units. In units with multipeaked tuning curves or clearly separated response areas, bicuculline application removed the inhibitory regions and created single-peaked tuning curves. The influence of bicuculline on the receptive field size was not significantly layer-specific but tended to be most pronounced in layers V and VI. In layer VI, we frequently found "silent" neurons that responded to sound only when GABAergic inhibition was antagonized. From the analysis of postembedding GABA immunocytochemistry, the proportion of GABAergic neurons was found to be maximal in layers I and V, and the number of GABAergic perisomatic puncta (axon terminals) on cell somata peaked in layer V.