In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase FA/glycogen synthase kinase 3 alpha (kinase FA/GSK-3 alpha) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase FA/GSK-3 alpha phosphorylates NF-M predominantly on serine residue. Three major tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser603-Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase FA/GSK-3 alpha. By using the same approach, we also identified the in vivo sites of Ser502-Pro, Ser506-Pro, and Ser666-Pro as the other three major sites in NF-M phosphorylated by kinase FA/GSK-3 alpha. Taken together, the results provide initial evidence that kinase FA/GSK-3 alpha may represent a physiologically relevant protein kinase involved in the vivo phosphorylation of NF-M. Because Ser502, Ser506, Ser603, and Ser666 are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that FA/GSK-3 alpha may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system.