Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser23 as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S1 (14RRVPTETRSSF24) and S2 (21RSSFSRVSGSPSSGFRSQSWS41), localized within the amino-terminal head domain region. Ser23 was identified as a phosphorylation site on the 32P-labeled α-chymotryptic peptide that carried >80% of the 32P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S1 and S2 were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser23 was the major site of phosphorylation for protein kinase A on both S1 and S2 peptides. Low levels of incorporation of 32P-phosphates into Ser22, Ser28, and Ser32 by protein kinase A were also observed. Protein kinase C incorporated 32P-phosphates into Ser22, Ser23, Ser25, Ser28, Ser32, and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S1 and that ions with up to four phosphates were detected on peptide S2. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser23 is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser23 on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.