To quantify the serotonin innervation in adult rat hippocampus, serotonin axon terminals (varicosities) were uptake-labeled for light microscope radioautography in whole hemisphere slices incubated with 1 microM [3H]serotonin. The labeled varicosities were visualized as small aggregates of silver grains and counted with the aid of an image analysis system across all layers in representative sectors of subiculum, Ammon's horn (CA1, CA3-a, CA3-b) and dentate gyrus (medial blade, crest and lateral blade). Counts were obtained in six rats at three equidistant horizontal levels from the ventral two-thirds of the hippocampus. After double correction for duration of radioautographic exposure and section thickness, and measurement of the mean diameter of labeled varicosities in electron microscope radioautographs, the results were expressed in number of varicosities per mm3 of tissue. The overall density of hippocampal serotonin innervation was thus evaluated at 2.7 x 10(6) varicosities per mm3, and appeared significantly higher in subiculum (3.6 x 10(6)) and Ammon's horn (3.1 x 10(6)) than in dentate gyrus (2.2 x 10(6)). Subiculum and dentate gyrus-crest (2.0 x 10(6)) had the highest and lowest regional densities. There was a marked heterogeneity also in terms of laminar distribution. For example, the stratum moleculare of subiculum and CA1, and the stratum oriens of CA3 (5.2 x 10(6)) varicosities in CA3-a), showed much higher values than the pyramidal cell layer (0.7, 1.1 and 0.7 x 10(6) in CA1, CA3-a and CA3-b, respectively). Similarly, the granular layer of dentate gyrus had a much lower density (1.1 x 10(6)) than did the molecular (2.8 x 10(6)) and the polymorph layer (2.4 x 10(6)). From these data, it was possible to evaluate the mean endogenous amine content per hippocampal serotonin varicosity (0.05-0.07 fg), and the average number of serotonin varicosities per hippocampal neuron in both CA3 (130) and dentate gyrus (20-35). In the context of current data on the distribution of serotonin receptors and diverse actions of serotonin at the cellular level in hippocampus, such quantified information provides new insights on some basic properties of serotonin in this part of the brain.