Staphylococcus aureushas been isolated at an increasing rate from infants’ stools during the last decades, but it is not known whether this species can colonize and persist in the intestinal microflora. To investigate this, 49 Swedish infants were followed prospectively from birth until 12 months of age.S. aureuswas identified in a rectal swab obtained 3 d after delivery and in quantitative cultures of fecal samples collected at 1, 2, 4, and 8 weeks and at 6 and 12 months of age. A random amplified polymorphic DNA (RAPD) method was developed to distinguish individualS. aureusstrains from one another and the strains were tested for production of enterotoxins A-D and TSST-1. By 3 days of age, 16% of infants hadS. aureusin their intestines, which increased to 73% by 2-6 months, whereafter it decreased slightly to 53%. At the same timeS. aureuspopulation counts in colonized infants declined from an average 106.8CFU/g feces during the first months of life to 104.0CFU/g feces by 12 months. Colonized infants usually harbored one or twoS. aureusstrains in their microflora for long periods of time. Few strains were transient passengers and the median time of persistence ofS. aureusstrains in the microflora was several months. Of the 75S. aureusstrains identified, 43% produced one or more toxins: 13% SEA, 7% SEB, 23% SEC, 4% SED, and 11% TSST-1. Altogether, 47% of the investigated infants were colonized by a toxin-producingS. aureusduring their first year of life. Despite this they were apparently healthy and did not have more gastrointestinal problems than noncolonized infants. This report is the first to show thatS. aureusmay be a resident member of the normal intestinal microflora in infancy.