The novel thiol-group-selective bifunctional 18F-labeling agent N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM) has been developed. The bifunctional labeling precursor N-(6-aminoxyhexyl)maleimide containing a thiol-reactive maleimide group and a carbonyl-group-reactive aminooxy group was prepared in only three steps in a total chemical yield of 59%. Subsequent radiolabeling with 4-[18F]fluorobenzaldehyde gave the bifunctional 18F-labeling agent [18F]FBAM in a radiochemical yield of 29%. In a typical experiment, 3.88 GBq of [18F]fluoride could be converted into 723 MBq of [18F]FBAM within 69 min. Conjugation of [18F]FBAM with thiol groups was exemplified with the cysteine-containing tripeptide glutathione and with various apolipoproteins of human low-density lipoprotein (LDL) subfractions. The latter was evaluated with respect to the uptake of [18F]FBAM-LDL subfractions in human hepatoma cells (HepG2) in vitro. In vivo biodistribution studies in rats revealed high stability for [18F]FBAM-LDL subfractions. Moreover, the metabolic fate of [18F]FBAM-LDL subfractions in vivo was delineated by dynamic positron emission tomography studies using a dedicated small animal tomograph. Data were compared to former studies that used the NH2-reactive 18F-labeling agent N-succinimidyl-4-[18F]fluorobenzoate. The compound [18F]FBAM can be considered as an excellent prosthetic group for the selective and mild 18F labeling of thiol-group-containing biomolecules suitable for subsequent investigations in vitro and in vivo.