With conventional immunocytochemical techniques on fixed tissue, antibodies which recognize highly phosphorylated neurofilament proteins strongly label axons, but often react poorly with perikaryal neurofilaments. The reactivity of one such antibody, SMI31, with neurofilaments in vivo has been investigated by microinjecting purified SMI31 into large neurons in living cultures of embryonic mouse spinal cord. Microinjected SMI31 (SMI31I) labeled perikarya and dendrites in a fibrillar pattern indistinguishable from that of microinjected SMI32 (SMI32I), which labels hypophosphorylated neurofilaments of perikarya and dendrites in fixed tissue. SMI31 also labeled perikarya and dendrites when applied to whole unfixed cultures after extraction with 1% Triton X-100 or to cultures fixed in acetone after Triton-extraction, but prior to exposure to primary antibody. SMI31 labeled mainly axons when applied after fixation with acetone without Triton-extraction. Positive labeling of neurofilaments and various inclusions in neuronal somata with antibodies against highly phosphorylated neurofilaments has been described in a number of neurotoxic and neurodegenerative diseases and after neuronal injury. The results of this study indicate that explanations other than alterations in phosphorylation could account for these observations.