I have investigated the phosphorylation state of the medium molecular mass neurofilament protein (NF-M) along axonal neurofilaments. Cultured embryonic sensory neurons were treated with non-ionic detergent to cause the cytoskeletal polymers to splay apart from each other. Neurofilaments were visualized by double-label immunofluorescence microscopy and the proportion of their length that stained with various NF-M antibodies was determined using digital image analysis techniques. Monoclonal antibody RMO255, which binds to NF-M independently of phosphorylation state, stained an average of 98% of the neurofilament length. In contrast, monoclonal antibody RMO55, which binds specifically to a phosphorylated epitope on NF-M, stained some neurofilaments completely, some not at all, and some along part of their length. These partly stained neurofilaments exhibited single or multiple discrete segments of staining along their length separated by segments that were unstained. The average proportion of the neurofilament length that stained with this antibody was lowest proximally (12-22%, n=3) and increased along the axon to reach a maximum distally (58-87%, n=3). A converse pattern (77-87% proximally and 2-9% distally, n=3) was observed for neurons stained with monoclonal antibody FNP7, which binds to specifically to a non-phosphorylated epitope in both NF-M and the high molecular mass neurofilament protein, NF-H. Analysis of the staining of individual neurofilaments revealed a bimodal frequency distribution in which neurofilaments were more likely to be phosphorylated along either all or none of their length than along part of their length. These observations indicate that: (a) phosphorylated and non-phosphorylated neurofilaments can coexist side-by-side in these axons, (b) neurofilaments can be composed of single or multiple contiguous phosphorylated and non-phosphorylated epitope domains along their length, (c) the proportion of the neurofilament length that is phosphorylated at these epitopes increases along the axon in a proximal-to-distal manner, and (d) the pattern of phosphorylation is non-random, generating populations of phosphorylated and non-phosphorylated neurofilaments and discrete phosphorylated and non-phosphorylated domains along individual neurofilaments.