Cows with Staphylococcus aureus mastitis received either one or two intramammary infusions containing 100,000 U penicillin G and 150 mg novobiocin. Milk and tissue samples were obtained from each quarter. Peak mean penicillin and novobiocin concentrations from antibiotic-positive tissue samples were .013 U/mg and .06 microgram/mg, respectively, for quarters treated once. Quarters treated twice had peak mean penicillin and novobiocin concentrations of .057 U/mg and .06 microgram/mg, respectively. Viable Staphylococcus aureus were isolated intermittently from milk and tissue samples of quarters positive for penicillin and novobiocin for both treatment groups. Histological analysis of mammary parenchyma demonstrated marked decreases in luminal area and increases in connective tissue area and leukocyte infiltration in S. aureus-infected quarters compared with uninfected controls, suggesting that reduction in milk collecting space and presence of inflammatory changes may be responsible for poor drug distribution.