We have previously reported that cells in cat areas 17 and 18 can show increases in response to non-optimal orientations or directions, commensurate with a loss of inhibition, during inactivation of laterally remote, visuotopically corresponding sites by iontophoresis of γ-aminobutyric acid (GABA). We now present anatomical evidence for inhibitory projections from inactivation sites to recording sites where ‘disinhibitory’ effects were elicited. We made microinjections of [3H]-nipecotic acid, which selectively exploits the GABA re-uptake mechanism, < 100 μm from recording sites where cells had shown either an increase in response to non-optimal orientations during inactivation of a cross-orientation site (n = 2) or an increase in response to the non-preferred direction during inactivation of an iso-orientation site with opposite direction preference (n = 5). Retrogradely labelled GABAergic neurons were detected autoradiographically and their distribution was reconstructed from series of horizontal sections. In every case, radiolabelled cells were found in the vicinity of the inactivation site (three to six within 150 μm). The injection and inactivation sites were located in layers II/III–IV and their horizontal separation ranged from 400 to 560 μm. In another experiment, iontophoresis of biocytin at an inactivation site in layer III labelled two large basket cells with terminals in close proximity to cross-orientation recording sites in layers II/III where disinhibitory effects on orientation tuning had been elicited. We argue that the inactivation of inhibitory projections from inactivation to recording sites made a major contribution to the observed effects by reducing the strength of inhibition during non-optimal stimulation in recurrently connected excitatory neurons presynaptic to a recorded cell. The results provide further evidence that cortical orientation tuning and direction selectivity are sharpened, respectively, by cross-orientation inhibition and iso-orientation inhibition between cells with opposite direction preferences.