In this study we characterized a bacteriocin, warnericin RB4, produced by Staphylococcus warneri RB4. Warnericin RB4 activity was completely inactivated by trypsin and actinase E. The activity was stable at 100 degrees C for 15 min, and had a pH range of 2 to 6. S. warneri R134 showed antibacterial activity against only Alicyclobacillus acidoterrestris, A. acidocaldarius, and Micrococcus luteus, among 34 bacterial species tested. The amino acid sequence of the purified bacteriocin contained 27 amino acid residues (K-K-K-S-G-V-I-P-X-V-X-H-D-X-H-M-N-X-F-Q-F-V-F-X-X-X-S). The molecular mass of the bacteriocin was estimated to be 2,958.2 Da by ESI-MS. These results show that the Warnericin RB4 exhibiting specific antibacterial activity against thermo-acidophiles, Alicyclobacillus spp., is a Nukacin ISK-1 or closely related bacteriocin, classified with class IA (Lacticin 481 types). This is the first report that Warnericin RB-4 is effective to inhibit the growth of causative microorganisms of spoilage in various acidic drinks. Warnericin RB4 might prove useful in fruit juices and fruit juice-containing drinks.