We sought to determine the impact of 17beta-estradiol throughout the hippocampal trisynaptic pathway and to investigate the afferent fiber systems within CA1 and CA3 in detail. To achieve this objective, we utilized multielectrode arrays to simultaneously record the field excitatory postsynaptic potentials from the CA1, dentate gyrus, and CA3 of rat hippocampal slices in the presence or absence of 100 pM 17beta-estradiol. We confirmed our earlier findings in CA1, where 17beta-estradiol significantly increased field excitatory postsynaptic potentials amplitude (20%+/-3%) and slope (22%+/-7%). 17beta-Estradiol significantly potentiated the field excitatory postsynaptic potentials in dentate gyrus, amplitude (15%+/-4%) and slope (17%+/-5), and in CA3, amplitude (15%+/-4%) and slope (19%+/-5%). Using a high-density multielectrode array, we sought to determine the source of potentiation in CA1 and CA3 by determining the impact of 17beta-estradiol on the apical afferents and the basal afferents within CA1 and on the mossy fibers and the associational/commissural fibers within CA3. In CA1, 17beta-estradiol induced a modest increase in the amplitude (7%+/-2%) and slope (9%+/-3%) following apical stimulation with similar magnitude of increase following basal stimulation amplitude (10%+/-2%) and slope (12%+/-3%). In CA3, 17beta-estradiol augmented the mossy fiber amplitude (15%+/-3%) and slope (18%+/-6%) and the associational/commissural fiber amplitude (31%+/-13%) and slope (40%+/-15%). These results indicate that 17beta-estradiol potentiated synaptic transmission in each subfield of the hippocampal slice, with the greatest magnitude of potentiation at the associational/commissural fibers in CA3. 17beta-Estradiol regulation of CA3 responses provides a novel site of 17beta-estradiol action that corresponds to the density of estrogen receptors within the hippocampus. The implications of 17beta-estradiol potentiation of the field potential in each of the hippocampal subfields and in particular CA3 associational/commissural fibers for memory function and clinical assessment are discussed.