The growth of Staphylococcus aureus occurs at a wide range of pH(5-10), while the optimal is pH 7.0-7.5. The molecular mechanism of such pH tolerant properties should be elucidated because the production of the virulence factors was greatly affected by environmental pH. The effect of pH shift on the composition of cytosolic proteins in S. aureus was examined. A protein with a molecular mass of 23 kDa was remarkably enhanced by a pH upshift from 7 to 10. This alkaline shock protein (ASP23) was isolated and purified by ion-exchange chromatography. The N-terminal sequences of the purified protein and the protease-digested peptides were analyzed. The 320-bp DNA fragment that was designed from the peptide analysis was amplified. Using the amplified fragment as a probe, the ASP gene, asp23, was cloned. The deduced primary sequence of ASP23 comprised 169 amino acids with a calculated molecular weight of 19,191. Northern analysis revealed that asp23 was positively regulated at the transcription level by alkaline shock. Homology search revealed that asp23 is a novel gene. Although the physiological role of ASP23 has yet to be further analysed, we suggest that ASP23 plays a key role in alkaline pH tolerance of S. aureus.