Inferior olivary (IO) neurons are electrically coupled through gap junctions and generate synchronous subthreshold oscillations of their membrane potential at a frequency of 1-10 Hz. Whereas the ionic mechanisms of these oscillatory responses are well understood, their origin and ensemble properties remain controversial. Here, the role of gap junctions in generating and synchronizing IO oscillations was examined by combining intracellular recordings with high-speed voltage-sensitive dye imaging in rat brain stem slices. Single cell responses and ensemble synchronized responses of IO neurons were compared in control conditions and in the presence of 18beta-glycyrrhetinic acid (18beta-GA), a pharmacological gap junction blocker. Under our experimental conditions, 18beta-GA had no adverse effects on intrinsic electroresponsive properties of IO neurons, other than the block of gap junction-dependent dye coupling and the resulting change in cells' passive properties. Application of 18beta-GA did not abolish single cell oscillations. Pharmacologically uncoupled IO neurons continued to oscillate with a frequency and amplitude that were similar to those recorded in control conditions. However, these oscillations were no longer synchronized across a population of IO neurons. Our optical recordings did not detect any clusters of synchronous oscillatory activity in the presence of the blocker. These results indicate that gap junctions are not necessary for generating subthreshold oscillations, rather, they are required for clustering of coherent oscillatory activity in the IO. The findings support the view that oscillatory properties of single IO neurons endow the system with important reset dynamics, while gap junctions are mainly required for synchronized neuronal ensemble activity.