Neurite outgrowth from larval frog spinal cord explants in tissue culture previously has been shown to be enhanced and oriented in the presence of mesenchymal limb target tissue. Evidence that suggests the limb mesenchyme is the source of a motor neuron growth factor has prompted consideration of a possible target dose relationship in eliciting spinal neurite growth. Whereas typical experimental paradigms incorporate a single target explant in cocultures of neural and target tissues, we have challenged spinal cord explants with dual limb targets. Both the spinal cord explants and mesenchymal limb tissue segments were derived from young stage V Rana pipiens tadpoles. In comparison to cultures with a single limb, the spinal neurite density in the presence of two limbs increased more than twofold. Accompanying the increase in neuritic quantity was a substantial increase in the average length of the neurites. Over a two-week culture period there was little change in the relative differences between the single and double target cultures. Even though the double target tissues were placed adjacent to one another on one side of the cord explant, greatly enhanced neurite growth occurred on the contralateral side as well. The dramatic increase in neurite outgrowth in the presence of an additional target tissue provides further evidence that undifferentiated mesenchymal limb tissue is the source of a spinal neurite growth-promoting factor that may act in a dose-dependent manner with implications for both motor axon growth regulation and regeneration.