We tested whether regeneration of transected rubrospinal tract (RST) axons is facilitated by a prolonged electrical stimulation of these axons. A peripheral nerve was grafted to the transected RST at the cervical level (C4/5) of adult rats, providing a permissive environment for regeneration of rubrospinal axons. Direct antidromic stimulation of the RST was applied immediately after grafting through a microwire inserted just rostral to the RST lesion, using a 1-h 20-Hz supramaximal stimulation protocol. Stimulation caused no direct damage to rubrospinal axons, and was sufficient to recruit the entire rubrospinal tract. In control animals that had a nerve graft and implanted microwire with no stimulation, there were 42.7 ± 10.2 rubrospinal neurons regenerated into the graft at 8 weeks, as assessed by retrograde labelling. In test animals that were stimulated there were 28.2 ± 7.4 backlabelled neurons, not significantly different from control, indicating that this stimulation did not improve the regenerative capacity of rubrospinal neurons. Furthermore, reverse-transcriptase polymerase chain reaction and in situ hybridization for brain-derived neurotrophic factor (BDNF) and/or growth-associated protein-43 (GAP-43) expression in rubrospinal neurons revealed no significant difference between stimulated and unstimulated groups at 48 h after injury, with either 1 or 8 h of stimulation. In summary, direct stimulation of the injured RST axons for the periods tested does not increase expression of GAP-43 and BDNF, and ultimately does not promote regeneration of these central nervous system axons.