We have previously demonstrated that both peripheral and central neurons from embryonic chick and newborn mouse can be maintained in a serum-free defined culture medium containing the appropriate neuronotrophic agent and the N1 supplement consisting of insulin, transferrin, putrescine, progesterone and selenite. In the present studies we have examined the short-term survival requirements of 8-day embryonic chick ciliary ganglion (CG) neurons. By comparing CG neuronal survival in our standard culture medium, Eagle's Basal Medium (EBM), with several other commercially available basal media, we have established that CG neurons also have specific requirements for pyruvate, serine and iron (Fe3+), in addition to their trophic factor (Ciliary Neuronotrophic Factor, CNTF) and the N1 supplement. The data suggest the existence of 3 subsets of CG neurons differing in their essential needs, namely: (1) those supported by glucose in the absence of pyruvate, (2) those requiring exogenous pyruvate but not serine or Fe3+, and (3) those which need pyruvate, serine and Fe3+. The minimal effective concentration of pyruvate could be decreased by a factor of 50 in the concurrent presence of serine and Fe3+. Serine was also a limiting element in the survival of some of these CG neurons. The Fe3+ concentration required by the same neurons was considerably diminished with the availability of transferrin, perhaps reflecting an increased Fe3+ transmembrane transport efficiency. Insulin was found to be the only N1 ingredient required for the survival of CG neurons. Insulin was a constant requirement for all 3 subsets of CG neurons, even when cultured in the total absence of glucose (but presence of pyruvate).