Previously we reported visualisation of a sub-population of fibroblast-like cells localised to neuromuscular junctions (NMJ), using polyclonal 2166-antibody (Court et al., 2003). We have now extended these observations by showing first, that the 2166 antibody probably recognises an unusual conformational form of tubulin. Second, immunocytochemical stains with CD34 antibody together with volume-rendered electron micrographs of a serially-sectioned NMJ from triangularis sterni (TS) muscles isolated from twelve mice killed by cervical dislocation (Home Office Schedule 1), showed extensive capping of motor endplates by the membranes of the 2166-positive junctional cells. Next, TS muscles were partially denervated by intercostal nerve section with Home Office authority in eight mice anaesthetised with ketamine/xylazine (respectively 100/10 mg/kg, IP). The mice were killed by cervical dislocation 1-6 days later. Muscles were isolated and prepared for whole-mount immunostaining. Within 24 hrs of nerve injury, 2166-positive cells had extended many fine cellular processes and this coincided with strong expression of tenascin C, a molecule implicated in the induction of motor nerve sprouting (Cifuentes-Diaz et al., 1998). By 3 days, terminal Schwann cells became reactive (nestin positive) and extended sprouts along 2166-positive cellular processes, pervading the tenascin-C positive extracellular matrix. To test whether the 2166-positive cells play an instructive role in activating Schwann cells, we compared their responses to denervation with the effects of muscle paralysis. Botulinum toxin (type A; 2 ng/kg, SQ) was injected into the interstitial spaces internal to intercostal muscles of six ketamine/xylazine-anaesthetised mice and the muscles examined after killing the mice 1-6 days later. After 1 day, junctional fibroblasts sprouted to a level comparable to that in denervated muscles. The spread of 2166-positive cells continued over the following 5 days. However, terminal Schwann cells neither became nestin positive nor did they sprout within 6 days of TS muscle paralysis. Together, these data suggest that the 2166-positive NMJ cells play a permissive role in triggering Schwann cell and motor nerve terminal sprouting. Stimuli independent of those that activate the junctional fibroblasts are required to activate terminal Schwann cells and induce them to sprout.