Fusion experiments to produce monoclonal antibodies (mAbs) to surface proteins of the advanced third-stage larva (aL3) of Gnathostoma spinigerum were attempted in this study. It was demonstrated that the sera from Balb/c mice infected with live aL3, but not those immunized with crude larval extracts, immunoprecipitated a 25-kDa surface protein from the extract of 125I-labeled aL3. Hybridomas derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissues of aL3 including the esophagus, intestine, muscle and cuticle by immunofluores-cence assay (IFA). Twelve of 17 hybridomas showed fluorescent staining with the cuticle only. None of these cuticle-positive hybridoma lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. After two rounds of limiting dilution, mAbs derived from one of the hybridoma lines still strongly reacted with the cuticle by IFA. However, these mAbs did not react with any protein in the extract of aL3 metabolically labeled with [35S]methionine. In the Western blot analysis, the mAbs recognized proteins of molecular weights ranging from 55-96 kDa with prominent bands at 75 and 85 kDa. Four more fusion experiments resulted in several hybridomas producing antibodies that reacted with the 25-kDa surface protein by immunoprecipi-tation. These hybridomas later became unstable and discontinued secreting the specific antibodies.