We compared the survival requirements of early- and late-born motoneurons from E5 chicken spinal cord. Density gradient centrifugation followed by immunopanning using SC1 antibody allowed us to purify two size classes of motoneuron. Large motoneurons retained by 6.8% metrizamide were shown by BrdU labeling in ovo to be born on average 1.5 d earlier than the small motoneurons recovered from the metrizamide pellet. Large motoneurons were both biochemically and functionally more mature: they expressed higher levels of choline acetyltransferase and low-affinity neurotrophin receptor, and had an acute requirement for trophic support from muscle-derived factors. After 24 hr in culture in basal medium, all early-born motoneurons died, whereas 60% of late-born motoneurons survived. Small motoneurons can develop into large motoneurons in ovo, suggesting that they represent a general transitional stage in motoneuron development. Our results suggest that a defined period elapses between birth of a motoneuron and its acquisition of trophic dependence, possibly corresponding to the time required for target innervation. This property may have important consequences for the timing and regulation of developmental motoneuron death.