Monoclonal antibody 4F4, which was raised against a cell suspension of embryonic rat forebrain, reacts with acidic glycolipids and several high-molecular-weight glycoproteins in rodent brain. The major reactive glycolipid is maximally expressed at Embryonic Day 15 (E15) and is no longer detectable at Postnatal Day 14 (P14) in the rat. 4F4 antibody reacts with a glucuronic acid- and sulfate-containing lipid isolated from human sciatic nerve as well as with lipids from mouse and rat embryonic brain tissue. Although the glycolipid disappears postnatally, the immunoreactive glycoproteins continue to be expressed in brain until adulthood. Both sciatic nerve and embryonic brain glycolipids are hydrolyzed by glucuronidase/sulfatase treatment but are insensitive to all other glycosidases tested. In addition, the observed 4F4 reactivity with extracted glycolipids, glycoproteins, and tissue sections of embryonic brain is identical to the reactivity demonstrated by HNK-1 antibodies. Immunocytochemical studies in developing brain showed stage-specific distribution of this carbohydrate antigen. At E10 in the mouse, immunoreactivity is associated with the mantle layer of the neural tube. At E15 in the cortex, the most intense staining is associated with the molecular layer and the subplate, and weaker staining is seen in the intermediate zone and cortical plate, suggesting that the antigen is highly concentrated on postmigratory cells in the embryonic nervous system.