The distal nerve stump plays a central role in the regeneration of peripheral nerve but the relative importance of cellular and humoral factors is not clear. We have studied this question by freezing the tibial nerve distal to a crush lesion in cat. The importance of constituents from the near-nerve environment was assessed by modification of the contact between the tibial nerve and the environment. Silicone cuffs, containing electrodes for electrophysiological assessment of nerve regeneration, were placed around the tibial nerve distal to the crush site. The interaction between long acellular frozen nerve segments (ANS) and the near-nerve environment was ascertained by breaching the silicone cuff to allow access of cellular or humoral components. Tibial nerves were crushed and frozen for 40 mm and enclosed in nerve cuffs with 0.45-microm holes or 2.0-mm holes to allow access of humoral factors or tissue ingrowth, respectively. In a second set of experiments, tibial nerves were crushed and either frozen for 20+20 mm, leaving a 10 mm segment with viable cells in the center (stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable nerve segment. The regeneration was monitored electrophysiologically by implanted electrodes and after 2 months the nerves were investigated by light and electron microscopy. The results indicate that soluble substances in the near-nerve environment, such as nutrients, oxygen or tropic substances did not exert any independent beneficial effect on the outgrowing axons. However, phagocytic cells entering the acellular segment from the near-nerve environment were crucial for axonal outgrowth in long ANS.