The in vivo post-targeting of tumor by means of anti-carcinoembryonic-antigen (CEA) monoclonal antibodies (MAbs) and the avidin-biotin three-step system was tested by immunoscintigraphy and radioimmunoguided surgery (RIGS) in six patients with primary or recurrent rectal cancer. The patients were preoperatively injected with 1 mg of FO23C5 (anti-CEA) and/or B72.3 anti-tumor-associated glycoprotein (TAG-72) biotinylated MAb; after 24 hours, 1 mg of avidin was administered, and, after a further 24 hours, biotin labeled to Indium-111 (In111) was injected. Preoperative imaging was obtained by means of a gamma camera, and a portable gamma-detecting probe (Neoprobe 1000) was intraoperatively used to count tumor and surrounding normal tissue. Eight tumor sites were localized in the six patients. Four lesions were identified preoperatively and six intraoperatively with a mean tumor-to-normal-tissue (T/NT) ratio of 1:8. This method allowed preoperative scintigraphy and intraoperative radioimmunodetection to be performed with a single radioactive compound injection of biotin labeled to In111 within few days before surgery.