A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion and fenthion. Three haptens with different spacer-arms were synthesized. The haptens were conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens. Rabbits were immunized with the immunogens and six polyclonal antisera were produced and screened against each of the coating antigens using competitive indirect enzyme-linked immunosorbent assay for selecting the proper antiserum. The effect of hapten heterology on immunoassay sensitivity was also studied. The antibody-antigen combination with the most selectivity for malathion was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC(50) values, under optimum conditions, were estimated to be 30.1microgL(-1)for malathion, 28.9microgL(-1) for dimethoate, 88.3microgL(-1) for phenthoate, 159.7microgL(-1) for phosmet, 191.7microgL(-1) for methidathion, 324.0microgL(-1) for fenitrothion, 483.9microgL(-1) for methyl parathion, and 788.9microgL(-1) for fenthion. Recoveries of malathion, dimethoate, phenthoate, phosmet and methidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%. This assay can be used in monitoring studies for the multi-residue determination of O,O-dimethyl organophosphorus pesticides.