Two families of carboxylic acid derivatives of the herbicides atrazine and simazine were synthesized for use as haptens in the development of immunoassays. One family was made by using monosubstituted (alky1amino)cyanuric chlorides and a variety of w-amino acids, giving spacers of varying lengths attached to one secondary amino group. The second family resulted from the replacement of the 2-chloro group of atrazine or simazine with 3-mercaptopropanoic acid. These haptens were conjugated to carrier proteins via N-hydroxysuccinimide active esters to make immunogens and ELISA antigens. Mono(alky1amino)cyanuric chlorides were also conjugated directly to bovine serum albumin and thyroglobulin under physiological conditions. ELISA and competitive inhibition ELISA results demonstrated that antibodies from immunized rabbits bound all of the conjugated and free s-triazines tested, including the parent compounds atrazine and simazine. The degree of binding was dependent on the N-alkyl groups present and on the length and position of attachment of the spacer arm. These results illustrate the usefulness of this synthetic approach in easily producing immunogenic haptens for a class of compounds that are difficult to work with because of their low solubility.The s-triazine herbicides are among the most commonly detected pesticides in water (Muir et al., 1978; Glotfelty e t al., 1984;Ervin and Kittleson, 1988;LeMasters and Doyle, 1989). This observation is due to a combination of factors, including widespread and heavy use, chemical and biological stability, and mobility in water. From 1983 through 1987 over 3 000 000 pounds of simazine and atrazine (Figure 1; 3a and 3b) were used in California alone (California Department of Food and Agriculture, [1983][1984][1985][1986][1987]. For these reasons, the State of California considers them as prime indicator compounds of groundwater contamination. Of additional concern is the poorly understood potential of the s-triazines and their nitroso and other derivatives for teratogenic, mutagenic, and carcinogenic effects (Janzowski et al., 1980; Waters et al., 1981). Thus, a rapid and inexpensive assay is needed to allow rapid screening of numerous surface and subsurface water samples, as well as for a variety of other analytical applications. The value of immunoassays for the analysis of pesticide residues has been well established (Hammock and Mumma, 1980; Newsome, 1986; Harrison e t al., 1988; Van Emon et al., 1989; Jung e t al., 1989). Recent literature includes descriptions of immunoassays for three specific s-triazine herbicides, atrazine (Huber, 1985; Bushway et al., 1988;Dunbar et al., 1985;Schlaeppi et al., 1989), cyanazine (Robotti et al., 1986), and terbutryne (Huber and Hock, 1985).Since the commercially important s-triazine herbicides constitute a large class of structurally related compounds that should be amenable to immunoassay as a group, we chose a synthetic approach that allows for the development of a wider range of antibody specificities and ultimate assay designs tha...