Spinal motoneurons from chick embryos were purified by retrograde transport and fluorescence-activated cell sorting. Growth conditions for motoneurons were studied, with experiments focused on the effects of conditioned media from chick myotubes, fibroblasts, and spinal cord dividing cells. Motoneurons rapidly extended neurites when plated onto polylysine-coated dishes that had been exposed to these conditioned media. Enzymatic analysis of the substratum-binding, neurite outgrowth-promoting activity from myotube-conditioned medium indicated that it contained heparan sulfate and protein. The neurite outgrowth-promoting activity sedimented as a peak centered at a density of 1.34 in associative cesium chloride gradients, and eluted near the void volume of a Sepharose CL-6B column. Inclusion of myotube conditioned medium in the culture medium of motoneurons also enhanced their survival over periods greater than 2 days in culture. This enhancement of survival could not be explained by myotube-conditioned medium providing motoneurons with a continuous supply of the neurite outgrowth-promoting activity. Media conditioned by spinal cord dividing cells and fibroblasts supported motoneuron survival to some extent, but this effect was not as great as that of myotube-conditioned medium.