Immunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatase-sensitive domains of the 200-kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity-purified anti-200-kd sera used to immunostain tissue labeled the neurofilament-associated 200-kd band in a phosphatase-sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice-cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200-kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross-linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200-kd neurofilament polypeptide are capable of associating with microtubules.