The possible co-existence of calretinin with other calcium binding proteins, parvalbumin and calbindin D28k, and with GABA, was studied in non-pyramidal cells of the rat dorsal hippocampal formation, using the mirror technique. The majority of the calretinin-containing neurons (83%) were found to be immunoreactive for GABA (79% in the dentate gyrus, 84% in the CA2-3, and 88% in the CA1 subfield). Most of the GABA-negative calretinin-immunoreactive neurons were located in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. Detailed analysis of the calretinin-immunoreactive cells of these subfields revealed that the two morphologically distinct types of calretinin neurons, i.e. the spiny and the spine-free cells, differ in their immunoreactivity for GABA. The overwhelming majority (92%) of the spine-free neurons were GABA-positive, whereas the immunoreactivity of spiny cells was ambiguous. At the sensitivity threshold of the immunocytochemical techniques used in the present study, most of the spiny cells (89%) had to be considered as GABA-negative, although the staining intensity in their cell bodies was somewhat above background level. Colchicine treatment resulted in a degeneration of calretinin-immunoreactive neurons; therefore, its effect on the GABA content of spiny neurons could not be evaluated. Nevertheless, the observations suggest that calretinin-containing neurons are heterogeneous both morphologically and neurochemically. Examination of the co-existence of calcium binding proteins revealed that none of the hippocampal cells contained both calretinin and parvalbumin in any regions of the hippocampal formation. Some overlap was detected between the calretinin- and the calbindin D28k-containing cell populations, 5.1% of the former and 6.2% of the latter were immunoreactive for both calcium binding proteins. This may be due to a small degree of cross-reactivity of the calbindin D28k antiserum with calretinin. Thus, our results demonstrate that the majority of calretinin-immunoreactive neurons are GABAergic and represent a subpopulation of non-pyramidal cells with no or only a negligible overlap with the subpopulations containing the other calcium binding proteins, parvalbumin and calbindin.