Neuron-specific enolase (NSE) has been increasingly recognized as a marker for neuroendocrine tumors including small cell carcinoma of the lung (SCCL). To prepare monoclonal antibodies (MAbs) specific for human NSE, we first developed a simple method of purifying NSE by direct chromatofocusing of a crude extract of human brain tissue. BALB/c mice were then immunized with our preparation of NSE, and MAbs against NSE were generated utilizing a hybridoma technique. The antibodies were screened against both NSE and non-neuronal enolase (NNE) by a solid-phase radioimmunoassay (SPRIA). After cloning and subcloning of hybridomas, two groups of anti-NSE MAbs were identified by SPRIA. One group reacted specifically with NSE but not with its isoenzyme NNE, irrespective of whether antigens were glutaraldehyde fixed or unfixed. A second group reacted with both NSE and NNE when the latter were glutaraldehyde fixed, but surprisingly with neither antigen in the absence of fixation. Group I antibodies were further characterized by immunoblotting, and by immunocytochemistry of normal brain and liver sections and sections of SCCL. The results further supported the specificity of group I antibodies for NSE. These MAbs have potential utility in the diagnosis and management of neuroendocrine tumors, and in further understanding the biology of NSE.