A lambda gt11 expression library was screened with a polyclonal antiserum directed against Wla Wolfgram protein which corresponds to the 2',3'-cyclic nucleotide 3'-phosphohydrolase. This antiserum also recognizes epitopes of the high protein subunit of neurofilaments (NF-H). An NF-H cDNA clone (pNF-H1: 1.7 kb) was isolated and characterized. Using pNF-H1 as a probe, a second cDNA clone (pNF-H2: 3 kb) was isolated from the lambda gt11 library. The two clones were sequenced and pNF-H2 was found to encode 80% of rat NF-H protein (coil-2 and C-terminal region). The C-terminal region contains an unusual sequence with stretches of repeats of Lys-Ser-Pro which represent possible phosphorylation sites. Specific localization in neurons of the corresponding mRNA was demonstrated by in situ hybridization using the pNF-H1 as a probe.