Highly stereospecific polyclonal antibodies (anti-CN) to cotinine (CN), a major metabolite of nicotine, were prepared from rabbit antisera to CN-linked keyhole limpet hemocyanin (KLH) by removing the antibodies to KLH and to its binding regions of CN. This was achieved by using immunoadsorbents consisting of insolubilized KLH onto CNBr-activated-Sepharose 4B. A new simple and rapid enzyme-linked immunosorbent assay (ELISA) of urinary CN was developed using the anti-CN. A brief outline of the method is as follows: CN-bovine thyroglobulin complex is coated onto wells of microtiter plates (1 ng/well), and then aliquots of urine samples or standard CN solutions were added followed by appropriate dilution of the anti-CN. The bound anti-CN antibodies are quantified spectrophotometrically with horseradish peroxidase-labelled anti-rabbit IgG and 2,2′-azino-di (3-ethylbenz- thiazoline)-6-sulfonic acid. Measurement of CN concentration in urine samples can be read off on a calibration curve drawn by using standard CN solutions. The standard curve ranged from 1 ng to 4 μ g /ml with an estimated lower limit of sensitivity of 7-8 ng/ml, resulting in within/between-assay CV (coefficient of variation) of lower than 10%. The method allowed one to assay more than 40 samples in duplicate by using just one plate, and is thus easily applicable to epidemiological investigations into exposure status to tobacco smoke.