The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for the polychlorinated dibenzo-p-dioxins is described. We previously reported the synthesis of haptens and generation of antibodies for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Antisera were screened with seven different coating antigens (hapten-protein conjugates), including trans-3-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methylpropeno ic acid (VII) and 5-(3,7,8-trichlorodibenzo-p-dioxin-2-yl)penta-trans,trans-2,4-dien oic acid (X). All inhibition screening and optimization studies were conducted using a less toxic surrogate standard for TCDD [2,3,7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] which responded similarly to 2,3,7,8-TCDD in the ELISA. The most sensitive assay from the screening studies [coating antigen VII-BSA, 0.1 microgram/mL, and antiserum 7598 (anti-X-LPH), 1:10,000] was further optimized and characterized. It exhibited an IC50 value of 12 pg/well (240 pg/mL), with working range from 2 to 240 pg/well (40 to 4800 pg/mL). The influence of various physical and chemical factors (time, solvent, detergent) was investigated. The optimized assay was then used to assess cross-reactivity by congeners of halogenated dioxins and related structures. DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to improved IC50 values.