Protein kinase activities associated with neurofilament (cAMP, cGMP, Ca2+-independent) were almost completely extracted by 0.8 M KC1. Two activities were separated by either sucrose gradient ultracentrifugation of phosphocellulose chromatography. One of them phosphorylates specifically neurofilament proteins and preferentially the triplet (200 kDa, 145 kDa and 68 kDa) but neither casein nor type IIA histone. The second activity was identified as casein kinase I and does not catalyze the phosphorylation of neurofilament protein.