Axonal segments transected from their cell body in vivo commonly undergo degeneration within 3–4 days (Wallerian degeneration). In lower vertebrates and invertebrates, however, some transected axonal segments survive for long periods ranging between 30 and 200 days. To circumvent the technical complications of studying the mechanisms underlying long-term survival of transected axons in vivo, we developed an in vitro system. We found previously that isolated axonal segments of cultured Aplysia neurons preserved their morphological integrity for an average duration of 7.6 days (range 2–14 days) and maintain their passive and excitable membrane properties. This survival occurred in the absence of de novo protein synthesis. In the present study we examined the influence of homologous neurons on the survival of transected axonal segments. We found that the average survival time of transected axons was doubled when co-cultured in physical contact with intact homologous neurons (average 15.3 days, range 2–27 days). During this period, the transected axons extended neurites, maintained normal passive and excitable membrane properties, formed electrotonic junctions with the intact neurons and maintained normal free intracellular Ca2+ levels. Consistent with these observations, electron micrographs of the transected axon revealed that the cytoskeletal elements of the axon appeared normal even 20 days after transection. In contrast, the mitochondria and smooth endoplasmic reticulum appeared damaged. As the prolonged survival was conditional on physical contact between the transected axon and the surrounding intact neurons, we suggest that the prolongation of survival time is promoted by the direct transfer of material from the intact neurons to the transected axon. However, co-culture of transected axons with homologous neurons did not fully mimic in vivo conditions, in which transected axons can survive for several months.