The light and electron microscopic localization of antigenic sites for a polyclonal antiserum directed against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), was examined in the hippocampal formation of the rat brain with a double-bridged peroxidase-antiperoxidase method. By light microscopy, the majority of varicose processes with intense TH-like immunoreactivity (LI) were contained in the hilus of the dentate gyrus (DG) and strata radiatum and lacunosum-moleculare of the CA3 region of the hippocampus. Only a few immunoreactive fibers were observed in the molecular and granule cell layers of the DG, in strata oriens and pyramidale of CA3, and in all layers of CA1. Electron microscopy confirmed that these labeled processes were primarily axons and axon terminals. Terminals with TH-LI were 0.4-1.1 micron in diameter and contained many small clear vesicles and from 0 to 3 larger dense-core vesicles. The number and types of associations formed by terminals with TH-LI were remarkably similar in the DG and hippocampus proper despite known differences in intrinsic cells and function. In both regions, the majority of terminals with TH-LI formed junctions on small (distal dendrites (52% of 112 in the DG; 67% of 116 in CA3) and dendritic spines (30% in the DG; 18% in CA3) that were both asymmetric and symmetric. In the DG, axosomatic junctions (2% of 112) were symmetric and occurred exclusively on the perikarya of granule cells, whereas junctions on large (proximal) dendrites were more numerous (16%), exhibited symmetric as well as asymmetric membrane specializations, and were of both granule (molecular layer) and nongranule (hilus) cell origin. In CA3, synaptic contacts on perikarya (5% of 116) and large (proximal) dendrites (10%) of both pyramidal cell and nonpyramidal cell origin were few and all symmetric. The distribution and types of synaptic associations formed by terminals with TH-LI in the CA1 region paralleled that seen in the CA3 region. In both the dentate and hippocampus proper, 10% of the terminals with TH-LI were observed closely apposed to unlabeled terminals that formed asymmetric synapses with dendrites and dendritic spines. In rare instances, TH-immunoreactive terminals were found in close association with the basement membrane of blood vessels, astrocytic processes, or with other unlabeled terminals not forming recognizable junctions. In addition TH-LI was occasionally detected within the cytoplasm of a minority of astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)