GABA immunoreactive neurons are present in all layers of cat AI and in the subjacent white matter; they are most numerous in layer II, the superficial half of layer III and layer IV. Double labeling immunofluorescence reveals that subpopulations of the GABA neurons are immunoreactive for the calcium binding proteins (CaBP), calbindin (28 kDa vitamin D-dependent calcium binding protein) and parvalbumin. Both proteins are present exclusively with GABA neurons but in subpopulations that are entirely separate. The two proteins together are present in approximately 70-75% of the GABA neurons; the largest group of GABA neurons displaying no CaBP immunoreactivity is in layers I-IIIA and VI. Calbindin immunoreactive neurons are present in two bands within cat AI: a superficial band, made up of numerous stained somata and processes, that includes layers II and IIIA and a deeper band, containing fewer neurons, that is coextensive with layer VI. Isolated calbindin somata are scattered between the two bands and very rarely in the subcortical white matter. Parvalbumin immunoreactive neurons are very densely packed in layers IIIB and layer IV, and include the majority of GABA neurons in layer IV; they are also numerous in layer VI. Parvalbumin immunoreactive neurons are much less numerous in layers II, IIIA and V and are absent from layer I. Light microscopic analyses suggest that the two subpopulations of GABA/CaBP neurons include several morphological types. In addition to the intrinsic somata and processes, numerous axons in white matter subjacent to AI are immunoreactive for either or both of the two proteins. These data demonstrate that cat AI is similar to other cortical areas in other species in possessing subpopulations of GABA neurons that express the CaBPs, calbindin and parvalbumin.