Hirschsprung's disease is characterized by the absence of enteric neurons in the myenteric and submucosal plexus and the presence of many unmyelinated axons, visible in ganglion like structures, in the aganglionic part of the bowel. In previous studies we showed that the immunoreactivity of a monoclonal antibody (2F11) specific for neurofilament proteins is increased in aganglionic bowel segments. We now investigated whether the increased neurofilament protein staining results from an increase in neurofilament protein immunoreactivity in the aganglionic segment or if it is also related to differences in the phosphorylation state of neurofilament proteins. Bowel resection specimens of patients with Hirschsprung's disease and control patients were investigated by immunohistochemical techniques using a panel of different monoclonal antibodies that are specific for neurofilament proteins and have well known reaction patterns against different phosphorylated epitopes present on two neurofilament proteins, the middle (NF-M) and the high (NF-H) molecular weight subunit. For comparison the specimens were also stained for acetylcholinesterase, neuron-specific enolase (NSE), S-100, and glial fibrillary acidic protein (GFAP). Immunostaining with this panel of antineurofilament-antibodies showed differences in the phosphorylation state of neurofilament proteins in the aganglionic and the ganglionic bowel segments of patients with Hirschsprung's disease. These changes involved the phosphorylation state of these proteins and the ratio of NF-H and NF-M in neurofilament proteins. Staining with NSE and S-100 showed no significant differences between Hirschsprung's disease patients and control patients. We surmise that during the ingrowth and differentiation of hypertrophic axons the composition of neurofilament proteins formed in the aganglionic bowel segment differs from the neurofilament proteins formed in the ganglionic and control bowel segments.(ABSTRACT TRUNCATED AT 250 WORDS)