The newly isolated alkalophilic Bacillus sp. B001 produced a high level of proteolytic activity (34277 U/mL) when grown in production medium, and a 28 kDa protease, designated AprB, was purified from the culture supernatant. Partial amino acid sequences were obtained by tandem mass spectrometry (MS/MS) and a pair of degenerate primers was developed to amplify a 467-bp genomic sequence. The observed and predicted amino acid sequences showed similarity with sequences of high-alkaline proteases from Bacillusclausii, Bacillusalcalophilus, and Bacillus lentus. High stability of AprB towards surfactants and oxidizing agents, an optimal pH of 10.0, and an optimal temperature of 60 degrees C suggest that this high-alkaline protease has potential applications for various industrial processes.