Neurofilament (NF)-enriched preparations from bovine spinal cord contain regulator-independent kinase activities that phosphorylate NF subunits as well as alpha-casein. CKI-7 (N-2-amino ethyl, 5-chloroisoquinoline, 8-sulfonamide), a specific inhibitor of casein kinase I (CKI), inhibits the phosphorylation of NF subunits in the neurofilament preparation. This inhibition occurs at a concentration range identical to concentrations where CKI-7 inhibits rabbit reticulocyte CKI phosphorylation of alpha-casein. Heparin, a specific inhibitor of casein kinase II (CKII), produced only 20% inhibition of 32P incorporation into NF subunits, and only at concentrations 5 to 10-fold higher than those required to inhibit CKII from reticulocytes. CKI from rabbit reticulocytes phosphorylated all three NF subunits (NF-H, NF-M and NF-L). Comparison of the tryptic phosphopeptide maps of NF-M, phosphorylated by the NF-associated kinase and CKI, indicates that the casein kinase I phosphorylates many of the peptides phosphorylated by the NF-associated kinase and this phosphorylation occurs at the carboxy terminal tail domain of the NF-M subunit. These studies suggest that the major independent kinase activity associated with NFs is CKI.