Although intercellular Ca2+ waves resemble spreading depression (SD) and occur in hippocampal organ cultures (HOTCs), SD has not been reported in these cultures. Accordingly, electrophysiological and Ca2+ imaging techniques were used to examine potential interrelations between Ca2+ waves and electrophysiological changes of SD. Our results show, for the first time, that HOTCs can support SD. Furthermore, two distinct Ca2+ waves were found to precede SD. The first traveled >100 micron/sec along the pyramidal cell dendritic layer. The second subsequently traveled mostly perpendicular to the pyramidal cell layer from CA3 (or CA1) but also in all directions from its area of initiation. This second, slower wave spread with the interstitial DC change of SD at millimeters per minute but always ahead of it by 6-16 sec. Heptanol, which uncouples gap junctions, blocked both of these Ca2+ waves and SD. Thus, two types of Ca2+ waves occur with the initiation and propagation of SD. The first might reflect interneuronal changes linked by gap junctions, whereas the second might stem from interastrocyte changes linked via similar connections. Because individual cells can be followed in space and time for protracted periods in HOTCs, this preparation may be ideal for studies designed to explore not only the mechanisms of SD but also the long-term consequences of SD, such as ischemic tolerance.