Injections of the fluorescent dyes Fast Blue or Granular Blue into either the hippocampus (volume approximately 50 nl) or the entorhinal area (100-150 nl) resulted in labeling by retrograde axonal transport of cells in the diagonal band of Broca (dbB) and the medial septum (MS). A large number (approximately 30%) of these cells contained glutamic acid decarboxylase (GAD)-like immunoreactivity, as determined by combined retrograde fluorescent tracing and GAD-immunohistochemistry. Not all GAD positive cells in the dbB and MS were labeled by fluorochromes in a single experiment. The GAD-stained and fluorochrome-containing cells were present at all rostro-caudal levels of the septum and appeared not to belong to any single morphological class of cells. Double staining experiments showed that the GAD-positive cells did not contain acetylcholinesterase reaction product. These findings provide evidence that a significant portion of the septo-hippocampal projection may utilize gamma-aminobutyric acid as a neurotransmitter.