In the hippocampus, estrogens increase dendritic arborization, long-term potentiation, neuroprotection, and participate in many functions related with learning, memory, and affective behaviors. The presence of both estrogen receptors alpha (ERα) and beta (ERβ) isoforms has been described in the hippocampus where they play different physiological roles. The aim of this study was to investigate, by using both techniques immunohistochemistry and Western Blot, the expression pattern of ERα and ERβ in the hippocampus of the rat along the estrous cycle. Western blot analysis was used to confirm the specificity of the antibodies used against ERα and ERβ and its relative content in the hippocampus. Results from immunohistochemical studies indicate that ERβ expression increased more than the ERα in CA1 and CA3 regions during all phases of the estrous cycle. ERβ immunoreactivity was mainly located in the nucleus and predominantly expressed in CA1 during estrous and metestrus, and in CA3 during diestrus. ERα was more abundant during estrous in comparison to other phases of the cycle in CA1 region, while it was more abundant during metestrus in CA3. Interestingly, the immunolocalization of ERα subtype was both cytoplasmic and nuclear. The overall results indicate that there is a differential expression, cellular localization, and distribution of both ER subtypes in CA1 and CA3 regions, suggesting different roles for these two receptors in the hippocampus along the estrous cycle.