Explants of Embryonic Day 18 (E18) rat entorhinal cortex were cocultured with Postnatal Day 6 mouse hippocampal slices to study CNS regeneration in vitro. The present report describes a double-labeling paradigm for quantitative analysis of the type of new growth seen in immature cultures. Entorhinal projection neurons in living static cocultures were retrogradely labeled with DiI or Texas red-dextran at 6 days in vitro and with dextran-FITC at 13 days. An intervening lesion to the entorhinodentate pathway was made at 8 days by replacing the hippocampal slices with fresh ones. About one-third of the new efferent entorhinal projections labeled with the second tracer could be characterized as true regeneration of axons from previously projecting entorhinal neurons by virtue of their being double labeled. The remaining two-thirds comprised new, late-arriving axons from previously nonprojecting cells. Earlier studies have shown that rat entorhinal axons will reinnervate hippocampal slices only if the lesions are made before 2-3 weeks in culture, equivalent to a postnatal age of 11-18 days. In a second series of experiments we tested whether treatment with trophic factors could overcome this age-related failure of regeneration characteristic of mature preparations. E18 explants were lesioned after 4 weeks in vitro and grown for a further 2 weeks in medium supplemented with either Schwann cell conditioned medium or acidic fibroblast growth factor plus heparin. A significant increase in outgrowth was seen in both cases, although the effects of each factor were not additive when they were applied in combination. These results show that our model of CNS lesions in vitro can be used to assess the effectiveness of growth factors in ameliorating the decline in regenerative ability with increasing developmental age.