Theta oscillations (4-12 Hz) are associated with learning and memory and are found in the hippocampus and the entorhinal cortex (EC). The spatio-temporal organization of rhythmic activity in the hippocampal-EC complex was investigated in vitro. The voltage sensitive absorption dye NK3630 was used to record the changes in aggregated membrane voltage simultaneously from the neuronal networks involved. Oscillatory activity at 7.0 Hz (range, 5.8-8.2) was induced in the slice with the muscarinic agonist carbachol (75-100 microM) in the presence of bicuculline (5 microM). Time relations between all recording sites were analyzed using cross-correlation functions which revealed systematic phase shifts in the theta oscillation recorded from the different entorhinal and hippocampal subregions. These phase shifts could be interpreted as propagation delays. The oscillation propagates over the slice in a characteristic spatio-temporal sequence, where the entorhinal cortex leads, followed by the subiculum and then the dentate gyrus (DG), to finally reach the CA3 and the CA1 area. The delay from dentate gyrus to the CA3 area was 12.4 +/- 1.1 ms (mean +/- s.e.m.) and from the CA3 to the CA1 region it was 10.9 +/- 1.9 ms. The propagation delays between the hippocampal subregions resemble the latencies of electrically evoked responses in the same subregions. Removing the entorhinal cortex from the slice changed the spatiotemporal pattern into a more clustered pattern with higher local synchrony. We conclude that in the slice, carbachol-induced theta oscillations are initiated in the entorhinal cortex. The EC could serve to control the information flow through the neuronal network in the subregions of the hippocampus by synchronizing and/or entraining their responses to external inputs.