The effect of media conditioned by muscle cells on the development in vitro of chicken spinal neurons was studied. Neural tube cells of 4.5-day chicken embryos were dissociated after trypsinization and cultured in serum-free minimum essential medium conditioned for 4 days over cultures of fused chicken myotubes. After 20 hr in conditioned medium (protein concentration, 10--50 microgram/ml), about 50% of surviving cells had extended neurites, whereas in cultures in nonconditioned medium this value was about 10%. The active factor(s) in conditioned medium is macromolecular and its activity was completely destroyed by incubation with trypsin. Concentrated samples of conditioned medium were analyzed by gel filtration on columns of Sepharose CL-6B. The activity was recovered in peaks with apparent molecular weights of 40,000 and 500,000 and at the exclusion volume of the column. Media conditioned neurite-promoting activity but at lower levels. No activity was detected in Nerve Growth Factor, insulin, fetal calf serum, or horse serum or in media conditioned by chicken lung, chicken heart, or C6 glioma cells.