The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB). The minimal inhibitory concentrations (MIC) of β-lactamase-positive strains in milk were 10–100-fold those in ISB, whereas the MIC of β-lactamase-negative strains in milk were some 10-fold those in ISB; β-lactamase production was induced by milk in β-lactamase-positive strains. Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria. It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth. In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.