Tolerance of Staphylococcus epidermidis in biofilms to killing by rifampin was correlated with limitation of bacterial growth in the biofilm state. Intact biofilm experienced a 0.62 log reduction when treated with 0.1 μg rifampin/ml for 4 h whereas the same treatment of exponential-phase planktonic cells produced a log reduction of 4.48. Stationary-phase planktonic cells were nearly as tolerant as intact biofilm cells, experiencing a 1.11 log reduction. Biofilm bacteria grew at only 10% of the maximum rate at which they grew on the same medium in planktonic culture. Killing was localized near the surface of the biofilm adjacent to the nutrient source, as revealed by staining with a respiratory dye. Increased nutrient concentration during antibiotic treatment enhanced killing of biofilm cells. Changing the oxygen tension in the gas phase above the biofilm during antibiotic treatment barely affected killing. It was hypothesized that the biofilm harbors significant numbers of stationary-phase-like cells in the nutrient-limited depths of the biofilm, and that these inactive cells are the ones that survive antibiotic challenge.