Microiontophoresis of γ-aminobutyric acid (GABA) was used to reversibly inactivate small sites of defined orientation/direction specificity in layers II-IV of cat area 17 while single cells were recorded in the same area at a horizontal distance of ~350–700 jam. We compared the effect of inactivating iso-orientation sites (where orientation preference was within 22.5 deg) and cross-orientation sites (where it differed by 45–90 deg) on orientation tuning and directionality. The influence of iso-orientation inactivation was tested in 33 cells, seven of which were subjected to alternate inactivation of two iso-orientation sites with opposite direction preference. Of the resulting 40 inactivations, only two (5%) caused significant changes in orientation tuning, whereas 26 (65%) elicited effects on directionality: namely, an increase or a decrease in response to a cell's preferred direction when its direction preference was the same as that at an inactivation site, and an increase in response to a cell's nonpreferred direction when its direction preference was opposite that at an inactivation site. It is argued that the decreases in response to the preferred direction reflected a reduction in the strength of intracortical iso-orientation excitatory connections, while the increases in response were due to the loss of iso-orientation inhibition. Of 35 cells subjected to cross-orientation inactivation, only six (17%) showed an effect on directionality, whereas 21 (60%) showed significant broadening of orientation tuning, with an increase in mean tuning width at half-height of 126%. The effects on orientation tuning were due to increases in response to nonoptimal orientations. Changes in directionality also resulted from increased responses (to preferred or nonpreferred directions) and were always accompanied by broadening of tuning. Thus, the effects of cross-orientation inactivation were presumably due to the loss of a cross-orientation inhibitory input that contributes mainly to orientation tuning by suppressing responses to nonoptimal orientations. Differential effects of iso-orientation and cross-orientation inactivation could be elicited in the same cell or in different cells from the same inactivation site. The results suggest the involvement of three different intracortical processes in the generation of orientation tuning and direction selectivity in area 17: (1) suppression of responses to nonoptimal orientations and directions as a result of cross-orientation inhibition and iso-orientation inhibition between cells with opposite direction preferences; (2) amplification of responses to optimal stimuli via iso-orientation excitatory connections; and (3) regulation of cortical amplification via iso-orientation inhibition.