Collateral sprouting is a form of neuronal plasticity observed in brain following injury. In order to establish an in vitro model of collateral sprouting, entorhino-hippocampal slice cultures were prepared from brain of C57BL/6 mouse pups (P1-4) and incubated for 14-16 days in vitro. Thereafter, entorhino-hippocampal fibers were cut and the outer molecular layer of the fascia dentata was denervated. At this age, entorhino-hippocampal fibers do not regenerate, as could be shown using anterograde tracing with Miniruby. Sprouting of associational mossy cell axons was monitored using calretinin-immunocytochemistry. Control and lesioned entorhino-hippocampal slices were studied at 1, 5, and 10 days postlesion. Whereas only the inner portion of the molecular layer was occupied by calretinin-positive mossy cell axons in controls and after 1 and 5 days postlesion, the entire width of the molecular layer was occupied by associational fibers by 10 days postlesion. Thus, robust sprouting of associational mossy cell axons occurs in response to entorhinal denervation in vitro. Using organotypic entorhino-hippocampal slices of genetically engineered mice, this sprouting model can be used to identify molecules involved in the regulation of sprouting following brain injury.