INTRODUCTION: Labeling single domain antibody fragments (sdAbs) with F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([F]RL-I), synthesized via a click reaction for labeling sdAbs with F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([F]SFETGMB; [F]RL-III) designed to address these limitations. METHODS: [F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[I]iodobenzoate ([I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[I]iodobenzoate (iso-[I]SGMIB). The F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [F]RL-III-5F7 also was performed. RESULTS: Radiochemical yields for the synthesis of Boc-[F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc-[F]RL-I. The overall radiochemical yields for the synthesis of [F]RL-III-2Rs15d and [F]RL-III-5F7 from aqueous [F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [F]RL-III-2Rs15d. Although different xenograft models were used, [F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [F]RL-III-5F7 in the intracranial model. CONCLUSIONS: Although higher radiochemical yields than that reported for [F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.