A general overview is presented of the changes in the genetic expression along a time curve through the first 20 min after acidification to pH 4.5 of exponentially growing cultures of the food pathogenic strain Staphylococcus aureus 50583. A newly developed method for statistical significance testing was used to detect significant gene expression responses. Most responses showed an increase or decrease from time zero to 10 min after acidification, and then generally a stabilization in expression level from 10 to 20 min. Increased urease activity appeared to be an important factor in the acid defence, along with proton excretion by NADH dehydrogenase and macromolecule repair mechanisms. Oxidative-stress responses, such as increased expression of thioredoxin genes and upregulation of pentose phosphate pathway genes to generate more reducing power, were also induced. A general reduction in the expression of genes encoding ribosomal proteins and genes involved in nucleotide synthesis, as well as fatty acid and lipoprotein metabolism, reflected the lowered growth rate after acidification. The pH shock did not appear to trigger major virulence responses or biofilm formation. Metal ion regulation and transport were affected by the acid shock, and production of several cofactors such as molybdopterin was increased. Many of the presented observations could be explained, while some represent still-unknown mechanisms. The patterns of regulation were confirmed by quantitative reverse transcriptase PCR (QRT-PCR). Together, these results showed the main responses of S. aureus and will be a good starting point for future, more specific, in-depth studies of specific gene responses that occur in conjunction with the acid-stress defence of S. aureus.