To obtain quantitative data on the distribution of the acetylcholine (ACh) innervation in the dorsal hippocampus of adult mouse (C57/B6) and rat (Sprague-Dawley), a semicomputerized method was used to measure the length of immunostained axons in hippocampal sections processed for light microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). The results could be expressed in density of axons (meters per mm3) for the different layers and regions of dorsal hippocampus (CA1, CA3, DG), and also in density of axon varicosities (millions per mm3), after having determined the average number of varicosities per unit length of ChAT-immunostained axon (4 varicosities/10 microm). In mouse, the mean regional densities of ACh innervation were thus measured at 13.9, 16.1, and 15.8 m of axons, for 5.6, 6.4, and 6.3 million varicosities per mm3 of tissue, in CA1, CA3, and DG, respectively. The values were comparable in rat, except for CA1, in which the densities were lower than in mouse by 40% in the stratum lacunosum, and 20% in the stratum radiatum. Otherwise, the laminar patterns of innervation were similar in the two species, the highest densities being found in the stratum lacunosum moleculare of CA3, pyramidale of both CA1 and CA3, and moleculare of DG. These quantitative data will be of particular interest to evaluate changes in mutant mice, or mice and rats subjected to experimental conditions affecting the cholinergic phenotype.