We have studied the distribution pattern and levels of expression of two estrogen receptor (ER) subtypes, ERalpha and ERbeta, in the normal adult (n = 10) and the aged (n = 10) female rat hippocampus with the objective to establish baseline data and the changes that occur during aging. Techniques including immunohistochemical localization, co-localization with double immunofluorescence and confocal microscopy, image analysis including neuronal counts/mm(2) area and measurements of optical density (OD) of immunoreactivity in immunoreactive neurons and Western blot analysis have been used. The results revealed ERalpha and ERbeta positive neurons in all subfields of the hippocampus with maximum presence in the stratum pyramidale of CA3. Some stained neurons in CA3 exhibited pyramidal neuron like morphological characteristics; such neurons were not found in CA1. All other immunoreactive neurons showed non-pyramidal neuron like morphological characteristics. Neuronal counts revealed a significant decrease in the number of immunoreactive neurons in CA3-CA1 of aged hippocampus. The percent decrease in counts of the immunoreactive neurons/mm(2) area in the aged rat (compared to the adult) was 78% for the ERalpha and 88% for the ERbeta (P < 0.001) in CA3. In CA1, it was 56% (P < 0.001) and 41% (P < 0.01) respectively. The OD of immunoreactivity was significantly decreased (P < 0.01) in CA3 but increased (P < 0.01) in the CA1 immunoreactive neurons. Western blot analysis also showed a significant decline (P < 0.01) in the levels of the ERalpha and ERbeta proteins in the aged hippocampus. Co-localization revealed that the two ER subtypes do co-exist in the same hippocampal neurons.