A newly isolated rat brain natriuretic peptide 45 (rBNP45) was synthesized using the 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase method. Tetrafluoroboric acid (HBF4) deprotection was successfully applied for this synthesis, while the conventional trifluoroacetic acid (TFA)-thioanisole method gave unsatisfactory results. The disulfide bond of rBNP45 was constructed by using the silyl chloride method within 10 min, which was extremely advantageous to avoid the formation of Met(O)-rBNP45. The chick rectum relaxant activity of the synthetic rBNP45 was three times as potent as that of alpha-rat atrial natriuretic peptide (alpha-rANP).