The cell membrane folate receptor is a potential molecular target for tumor-selective drug delivery. To probe structural requirements for folate receptor targeting with low molecular weight radiometal chelates, specifically the role of the amino acid fragment of folic acid (pteroylglutamic acid) in mediating targeting selectivity, the amide-linked conjugate pteroyl-NHCH2CH2OCH2CH2OCH2CH2NH-DTPA was prepared by a three-step procedure from pteroic acid, 2,2'-(ethylenedioxy)-bis(ethylamine),and t-Bu-protected DTPA. This conjugate, 1-{2-[2-[(2-(biscarboxymethyl-amino)ethyl)-carboxymethyl-amino]ethyl]-ca rboxymethyl-amino}-acetylamino-3,6-dioxa-8-pteroylamino-octane (1), was employed for synthesis of the corresponding In-111(III) radiopharmaceutical. Following intravenous administration to athymic mice, the In-111 complex of 1 was found to selectively localize in folate receptor-positive human KB tumor xenografts and to afford prolonged tumor retention of the In-111 radiolabel (5.4 +/- 0.8, 5.6 +/- 1.1, and 3.6 +/- 0.6% of the injected dose per gram of tumor at 1, 4, and 24 h, respectively). The observed tumor localization was effectively blocked by co-administration of folic acid with the In-111-1 complex, consistent with a folate receptor-mediated targeting process. In control studies, tumor targeting with this pteroic acid conjugate appears as effective as that seen using In-111-DTPA-folate, a radiopharmaceutical that has progressed to clinical trials for detection of folate receptor-expressing gynecological tumors.