The synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (amino carboxylic acid) attached at the pesticide thiophosphate group was simplified to a large extent. While the previous synthetic approach for this type of haptens requires seven steps, the present process involves only two steps. Using this process, five haptens of fenthion differing in spacer arm length (4–8 atoms) were synthesized and they were conjugated to bovine serum albumin and keyhole limpet hemocyanin to be used as immunogens. Rabbits were immunized with these hapten–protein conjugates for production of polyclonal antibodies against fenthion. The five haptens were conjugated to ovalbumin to be used as plate-coating antigens and twenty polyclonal antisera to the haptens were screened against each of the five coating antigens using noncompetitive and competitive indirect enzyme-linked immunosorbent assay (ELISA). The titer difference between the homologous and heterologous combinations was small, suggesting that heterology in spacer arm length is not important for the antigen recognition by antibodies. While the heterology in spacer length of the coating antigen had no significant effect on the sensitivity of ELISA, heterology in spacer structure of the coating antigen produced a remarkable improvement in the sensitivity of ELISA. © 2002 Elsevier Science B.V. All rights reserved.