The preparation of a new class of isothiocyanatopeptide bifunctional chelating agents, Boc-glycyl-L-(p-NCS)phenylalanylglycylglycine ethyl ester 1, N-(S-acetylmercaptoacetyl)-L-(p-NCS)phenylalanylglycylglycine ethyl ester 2, and N-(S-acetylmercaptoacetyl) glycyl-L-(p-NCS)phenylalanylglycine ethyl ester 3, starting from Boc-L-(p-nitro)phenylalanine 5 is described. The key intermediates, nitropeptides 7, 8 and 12, were prepared by standard DCC coupling and a deprotection procedure. The nitropeptides 7 and 12 on condensation with N-succinimidyl-S-acetylthioacetate provided a good yield of the corresponding S-(acetylmercaptoacetyl)nitropeptides 10 and 13, respectively. Catalytic hydrogenolysis of compounds 8, 10 and 13, followed by thiophosgenyation gave 28-95% yield of target products 1, 2 and 3, respectively. Compounds 1-3 were conjugated to the monoclonal antibody D612 reactive with human colon carcinoma using 12/1 to 60/1 ligand to antibody molar ratios, and the conjugates were labeled with 99mTc. The in vitro cell binding results of these immunoconjugates demonstrate that they retained their antibody binding specificity.