The production and evaluation of antibodies specific for 5-(2-bromo-E-ethenyl)-2'-deoxyuridine (BVDU) is described. Antibodies were raised against 5-(2-carboxy-E-ethenyl)-2'-deoxyuridine bovine albumin conjugates (CVDU20-BA and CVDU54-BA) and 5'-carboxy-propionyl-BVDU7-BA. The molar substitution ratio of the conjugates was determined by differential ultraviolet absorption or isotope dilution and compared to a newer method based upon the determination of unsubstituted protein residues. Usable antibody titers were obtained from all the New Zealand White rabbits immunized, but only one-third of the half-lop rabbits responded. The antisera were highly specific for BVDU and did not significantly cross-react with naturally occurring nucleosides, pyrimidines, or sugars. However, the lowest cross-reaction observed (0.21%) for the major metabolite bromovinyl uracil using antisera raised to CVDU-BA could not ensure adequate assay specificity under all conditions of use. An alternative approach using 5'-carboxy-propionyl-BVDU7-BA proved unsuccessful because the antisera could not discriminate between BVDU and bromovinyl uracil.