The middle molecular weight polypeptide of neurofilaments (NF-M) is modified posttranslationally by extensive phosphorylation. This modification is slow in mature neurons, requiring approximately 24-48 hr for completion and probably occurs outside of the cell soma (Bennett and DiLullo: J Cell Biol 100:1799, 1985c). Thus, NF-M synthesis and phosphorylation are separate events both temporally and spatially. Although it is known that NF-M is among the earliest neuron-specific gene products to be expressed during nervous system development, it is not known what the temporal relationship is between the initiation of NF-M translation and its phosphorylation. To address this question, we have produced an antiserum against the dephosphorylated form of NF-M (NF-M130) and have used this antiserum, together with a previously characterized antiserum against completely phosphorylated NF-M (NF-M160), in an immunohistochemical examination of neurogenesis and the initial period of neuronal differentiation in chick spinal cord. We found that 1) nonphosphorylated and partially phosphorylated NF-M cannot be detected prior to the completion of the terminal mitosis; 2) most postmitotic neuroblasts begin expressing NF-M as they commence migration, but do not contain the completely phosphorylated polypeptide until some time after completion of migration; and 3) those precursor cells of a subpopulation of neuroblasts that begin expressing completely phosphorylated NF-M during their terminal cell cycle (Bennett and DiLullo: Dev Biol 107:94, 1985a) contain no detectable nonphosphorylated or partially phosphorylated NF-M. These cells probably complete the phosphorylation step more rapidly than do mature neurons.