The neural cell-adhesion molecule (N-CAM) is detected as at least 3 related polypeptides generated by alternative splicing of a single gene. In vivo the 2 larger polypeptides are phosphorylated, but the smallest polypeptide, which lacks a cytoplasmic domain, is not. We have found that the 2 larger polypeptides are phosphorylated in vivo on several common phosphorylation sites. Furthermore, the largest polypeptide has additional sites, suggesting that some phosphorylation occurs in that portion of the intracellular region unique to it. In vitro N-CAM is not a substrate for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, II, or III, protein kinase C, or casein kinase II. However, we have isolated 2 protein kinases from mammalian and avian brain that phosphorylate rodent and chicken N-CAM. On the basis of their chromatographic behavior and substrate specificity, the 2 kinases are glycogen synthase kinase 3 (GSK-3) and casein kinase I (CK I). The 2 kinases phosphorylate N-CAM rapidly, to a high stoichiometry and with a low Km for N-CAM, suggesting that the phosphorylation of N-CAM by these kinases is physiologically relevant. Both enzymes phosphorylate the 2 larger N-CAM polypeptides in vitro in the cytoplasmic domain on threonyl residues that are phosphorylated to a low level in vivo. In addition, the threonyl residues are close to seryl residues phosphorylated to a high level in vivo. Prior phosphorylation at the in vivo sites appears to be a prerequisite for phosphorylation by GSK-3 and CK I. Taken together, the results suggest that N-CAM may be physiologically phosphorylated on 2 sets of interrelated sites, one demonstrable in vivo and one in vitro. Phosphorylation on the "in vivo" sites is resistant to dephosphorylation and may be constitutive, while phosphorylation on the "in vitro" sites is much more labile.