A 66 kd protein, pl 5.4, was purified from the Triton-insoluble fraction of rat spinal cord. This protein formed 10 nm filaments in vitro. The 66 kd protein was unique, although it shared homology with the 70 kd neurofilament protein (NF-L) and vimentin. An antiserum (anti-66) specific to the 66 kd protein did not cross-react with any of the neurofilament triplet proteins. In the spinal cord, anti-66 intensely stained the axons of the anterior and lateral columns. However, afferents from dorsal root ganglia and the efferents from the motoneurons were negative. In the cerebellum, anti-66 intensely stained most axons. The 66 kd protein was readily detectable in homogenates of forebrain, cerebellum, brainstem, and spinal cord, but was found only in trace amounts in adult sciatic nerves and was not found in extraneural tissues. The 66 kd protein constituted 0.5% of total protein in the spinal cord, whereas NF-L constituted about 1.5%.