Proteoglycans of developing chick brain were distinguished on the basis of reactivity with four well characterized antibody reagents (S103L, to the CS-rich domain; HNK-1, to 6-sulfated glucuronic acid; 1-C-3, to the HABr region and 5-D-4, to KS chains). One chondroitin sulfate proteoglycan reacted exclusively with S103L and 1-C-3 and not with the other two antibodies, hence is designated the S103L reactive brain CSPG. The other proteoglycan reacted exclusively with HNK-1 and 5-D-4 and not with S103L and 1-C-3, hence it is designated the HNK-1 reactive brain CSPG. In addition to these immunological distinctions, the S103L and HNK-1 CSPGs exhibited significant biochemical differences at both the protein and carbohydrate levels. Most interestingly, both CSPGs were found in all regions of the brain, and were expressed in a developmentally regulated pattern. The S103L CSPG was not detectable prior to embryonic day 7, increased to a maximum at day 13-15 and declined by day 20 in most brain regions examined. In contrast, the HNK-1 CSPG was present as early as embryonic day 4 and remained constant through hatching. Neuronal cultures established from embryonic day 6 (E6) cerebral hemispheres represent an in vitro paradigm that mimics in vivo neuronal development and differentiation. In this culture system we found that the expression of the S103L and HNK-1 CSPG followed a pattern similar to that observed in developing brain and further, that neurons are probably the sole source of S103L CSPG in cerebral cortex during neuroembryogenesis.