Staphylococcus aureus is a major cause of bovine mastitis. Since gene expression of many bacteria is known to be regulated by the environment, milk may play an important role in the regulation of the early steps in the pathogenesis of bovine mastitis by S. aureus. To get insight into the response of S. aureus to the milk environment, a Tn917-lacZ mutant library was generated and screened for genes specifically expressed during growth in milk. Twenty-eight mutants were identified and analysed. Four groups of genes were found, involved in cell-wall synthesis, nucleotide synthesis, transcriptional regulation and carbohydrate metabolism. A fifth group contained genes with hypothetical or unknown functions. Many of the genes identified belonged to biosynthetic pathways of S. aureus and other bacterial species which have also been shown to play a role in vivo as determined in murine infection models. Therefore, growth on milk may be an attractive model for the identification of genes preferentially expressed during bovine mastitis.