(1) Human neuron specific enolase (NSE) was purified from human brain tissue and used as an immunogen in the production of murine monoclonal antibodies. It was also used as a standard preparation of NSE in the screening of monoclonal antibodies and in the investigations of an enzyme-linked immunosorbent assay (ELISA) for NSE. (2) Four monoclonal antibodies were raised to NSE. These did not cross react with the alpha isoenzyme of enolase (NNE). Approximately 76% of positive well supernatants reacted with both NSE and NNE. (3) All four monoclonal antibodies reacted in an ELISA but only two of the four reacted on an immunoblot. (4) The monoclonal antibodies were purified by precipitation by ammonium sulphate and by Protein A-affinity column chromatography. The latter resulted in immunoglobulin which was pure by SDS-PAGE. (5) The purified monoclonal antibodies were labelled with horseradish peroxidase by the periodate oxidation method, with ¹²⁵I using the Chloramine T and the Bolton-Hunter methods, and with biotin. (6) Labelling with biotin was the most effective method. (7) The labelled monoclonal antibodies were used in the investigation of a sandwich ELISA for NSE. These experiments showed that high binding occurred with no antigen present. It was surmised that monoclonal antibody-monoclonal antibody binding may be occurring. (8) The high no-antigen control was investigated in a number of ways including altering the assay pH, the detergent concentration, the type of solid support and by using Fab fragments in the assay instead of whole molecule.