Motoneuron phenotype in the spinal cord is regulated by an intrinsic genetic program, extrinsic environmental signals and target-derived molecules. Axonal lesions trigger a phenotype switch to foster repair phenomena and axonal re-growth. We have investigated the influence of the long-term treatment with thyroid hormone and all trans retinol palmitate (RA) on motoneuron phenotype and spinal cord reaction to axotomy in adult male rats. Neurochemical markers, investigated by in situ hybridization and immunocytochemistry, included choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP) and neurotrophin low affinity receptor p75. Treatment was administered for 56 days and then mid-thigh sciatic axotomy was performed on a number of animals from each experimental groups; the rats were examined 9 days after surgery. The results indicate that: (1) Number and size of ChAT-immunoreactive neurons in the lumbar tract of the spinal cord was reduced in hypothyroid compared to control rats, whereas steady-state level of ChAT mRNA in labelled motoneurons failed to be modified by hypo and hyperthyroidism, but was increased by RA administration; (2) none of the administered treatments did alter CGRP mRNA level, whereas all of them influenced the axotomy-induced changes of motoneuron phenotype; (3) in hyperthyroid rats ChAT mRNA level of lumbar motoneurons not reduced homolateral to lesion while the number of ChAT-IR profiles was pronouncedly reduced; (4) up-regulation of p75 induced by peripheral nerve lesion was reduced in RA-treated rats. These data indicate that the motoneuron phenotype is regulated by transcription factors, which also play a role in phenotype switch regulation after axotomy.