Extracts of rat skeletal muscle contain neurotrophic factors which stimulate the development of choline acetyltransferase in embryonic day 14 rat spinal cord cultures. The trophic activity does not bind heparin-Sepharose or lectin affinity columns. However, mild acid treatment separates the trophic activity into soluble and insoluble fractions. The acid-insoluble activity has been purified 5000-fold to apparent homogeneity using preparative sodium dodecyl sulfate gel electrophoresis to achieve final purification. The purified factor migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent molecular mass of 20 kDa and a pI of 4.8. The activity and apparent molecular weight of the purified factor are unaltered by treatment with reducing agents or incubation in acidic conditions. Activity, however, is destroyed by heating or protease treatment. Thus, the factor appears to be a single polypeptide without significant levels of glycosylation or charge microheterogeneity. These results represent the first purification of a neurotrophic factor from skeletal muscle. The physical properties and amino acid composition of this factor differ from those of nerve growth factor and heparin-binding growth factors, as well as from the neurotrophic factor from heart cell conditioned medium which induces cholinergic development in sympathetic neurons.