The formation of the GABAergic septohippocampal projection was studied in vitro. Slice cultures of the septal complex from young postnatal rats were prepared and co-cultivated with hippocampal slices for up to four weeks. Then, the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin was injected into the septal culture and the labeled fibers were traced into the hippocampal culture. Some fibers were identified as originating from GABAergic septal cells by double-labeling with an antiserum against GABA using the postembedding immunogold procedure. Our results showed that double-labeled terminals of GABAergic septohippocampal neurons established symmetric synapses exclusively with GABA-positive dendrites in one out of five co-cultures, but also contacted numerous GABA-negative structures in the remaining four co-cultures. These findings, together with light microscopic data from sections double-stained for Phaseolus and parvalbumin, indicate that the high target selectivity of the GABAergic septohippocampal pathway for GABAergic interneurons in vivo is lost in most cases, at least under the present in vitro conditions. It is hypothesized that this may be due to an immaturity of the connection, the lack of axon-guiding factors or an expansion of the septohippocampal GABAergic fibers in the absence of many extrinsic afferents, including GABAergic fibers. The simultaneous occurrence of anterogradely labeled, but GABA-negative, septohippocampal terminals in the hippocampal target culture also suggests that the septohippocampal cholinergic projection developed in vitro, as was shown before in other studies. Since most septohippocampal neurons have to be axotomized for culture preparation, the present results indicate that GABAergic septohippocampal neurons from young postnatal rats survive axotomy and are capable of regenerating a septohippocampal projection, including the formation of characteristic GABAergic synapses on co-cultured hippocampal neurons. However, the characteristic target selectivity is rarely preserved.