A new tracer, N-5-[(18)F]fluoropentylmaleimide ([(18)F]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps ((18)F displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine, and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11-17% radiochemical yield (uncorrected), and specific activity of 20-49 GBq/μmol. [(18)F]FPenM showed comparable labeling efficiency with N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide ([(18)F]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys(40)]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and metabolic stability of the radiolabeled [(18)F]FPenM-[cys(40)]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [(18)F]FPenM-[cys(40)]-exendin-4, had high tumor uptake (20.32 ± 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was comparable to the imaging results with [(18)F]FBEM-[cys(40)]-exendin-4 reported by our group.