18F-labeling of proteins and peptides is important for positron emission tomography (PET). Although there are many methods for the labeling of proteins with (18)F, most of these are characterized by complicated procedures or low yields. Here, we report a novel and simple method which includes the preparation of [18F]fluorobenzaldehyde ([18F]FBA) and successive conjugation with hydrazinonicotinic acid-human serum albumin conjugate (HYNIC-HSA) via hydrazone formation. HYNIC-HSA, which can also be used for labeling with (99m)Tc, was prepared via reaction with N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) esters of HYNIC with HSA. No-carrier-added [18F]FBA was prepared by the nucleophilic substitution of [18F]fluoride to 4-trimethylammonium benzaldehyde triflate in the presence of tetrabutylammonium bicarbonate. [18F]FBA was purified by passing ion exchange cartridges (IC-H and QMA) and was adsorbed to a C18 Sep-Pak cartridge. The adsorbed [18F]FBA was then eluted with 50% ethanol. HYNIC-HSA was added to the solution and conjugated with [18F]FBA via hydrazone formation. 18F-HSA was purified with a PD10 column. Biodistribution of 18F-HSA, (99m)Tc-HSA, and [18F]FBA in mice were investigated at 10, 20, and 60 min after intravenous injection. The number of conjugated HYNIC molecules per HSA ranged from 5.2 to 23.2 depending on the reaction conditions. The labeling efficiency of 18F-FBA was 67 +/- 15.7%. The radiochemical purity after purification was over 99%. The conjugation efficiency of HYNIC-HSA with [18F]FBA was between 25% and 90%. The conjugation efficiency was observed to increase with increases in the number of conjugated HYNIC, the HYNIC-HSA concentration, or temperature. 18F-HSA exhibited a biodistribution pattern similar to that of (99m)Tc-HSA while [18F]FBA showed much lower blood activity than that of 18F-HSA and (99m)Tc-HSA. We concluded that 18F-HSA was successfully labeled using a novel method which involves hydrazone formation between [18F]FBA and HYNIC-HSA. This method can be applied to the 18F-labeling of other proteins or peptides.