The effect of Staphylococcus aureus on detachment of bovine mammary epithelial cells in culture was examined. Mammary epithelial cells became detached from fresh monolayers following a 3 h incubation in the presence of Staph. aureus M60. Two different procedures indicated that cell detachment coincided with the S-phase of the cell cycle. The roles of proteinases, toxins and Ca availability in inducing cell detachment were examined. Addition of the proteinase inhibitor phenyl-methylsulphonyl fluoride (1 mM) to the culture medium prevented cell detachment. Addition of a combination of purified staphylococcal proteinases XVI and XVII-B to the culture medium of mammary epithelial cells induced cell detachment in the absence of Staph. aureus. Cell detachment may be caused by a staphylococcal proteinase. However, addition of Ca (10 mM) to the culture medium abolished Staph. aureus-induced cell detachment, despite the fact that proteinase activity was still apparently present. Isogenic mutants of Staph. aureus M60, expressing either ± or β toxins but not both, induced cell detachment, but to a lesser extent than the wild type. Thus, Ca and toxins play some role during cell detachment. Clones established from detached cells that were washed and replated showed the same susceptibility to Staph. aureus-induced cell detachment as the parental cells. This indicated that there is no subclone of mammary epithelial cells more sensitive to this effect.