We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.