The distribution of galanin-like immunoreactivity in the rat hippocampal formation (hippocampus and dentate gyrus) was studied and its origins were determined using various lesioning techniques. Special reference was made to the known cholinergic and noradrenergic hippocampal inputs from the septum-basal forebrain complex and locus coeruleus, both of which have previously been shown to co-contain galanin-like immunoreactivity at the cell body level. Galanin-immunoreactive fibers in the hippocampal formation were of at least three different morphological types: (1) Fine, slender, faintly immunoreactive fibers were seen throughout the hippocampal formation. (2) A strongly fluorescent varicose fiber population was observed mainly in the strata radiatum and oriens of the ventral CA3 region. (3) A population of fine, faint puncta was seen within the granule and pyramidal cell layers throughout the hippocampal formation. Knife cut lesions of the dorsal afferent pathways resulted in almost complete disappearance of all fiber types, except for the ventral fine fibers. Lesions of the fimbria affected mainly the coarse and punctate fiber types, while lesions of the supracallosal striae depleted mainly the fine fibers. Cuts anterior and ventral to the hippocampal formation caused a decrease in ventral fine fibers. Furthermore, lesions of the dorsal bundle caused an almost complete disappearance of the fine fibers in all regions of the hippocampal formation. Neurotoxin lesions of the diagonal band/septal complex resulted in decreases in faintly immunoreactive puncta within the granule cell layer and adjacent fine fibers. It is concluded that most fine galanin-positive fibers originate in the lower brain stem, presumably the locus coeruleus, and appear to reach the hippocampal formation primarily through the supracallosal striae and the ventral route. The fimbria seems to contain a large proportion of the fibers giving rise to the coarse strongly fluorescent innervation, which appears to originate rostral to the pons. The galanin-immunoreactive fibers originating in cholinergic somata of the diagonal band, medial septal nuclei, previously shown to project to the hippocampal formation, seem to give rise to faintly labeled puncta within the granule and pyramidal cell layers, and to a small proportion of the fine fibers bordering the cell layers, as revealed by immunohistochemistry using our antibody.(ABSTRACT TRUNCATED AT 400 WORDS)