The aerobic bacterial flora of the top 25 layers of the stratum corneum of normal human skin was characterized by sampling glabrous skin with contact plates and analyzing plasmid patterns of coagulase-negative staphylococci (SCN) by agarose gel electrophoresis. The number of colonies of SCN on the skin surface at 12 sites varied from 14 to 838. Removal of five keratinized layers by sequential stripping with cellophane tape reduced the number of colonies by 80% (median; range, 42%-91%). Counts remained constant during removal of 20 additional layers. SCN with six different plasmid patterns were identified at a site on the skin surface. After removal of 25 layers, colonies with a single pattern were clustered in one quadrant of the site. The site was sterilized and covered with a sterile dressing for 18 h. Colonies reappeared in the same quadrant of the site; six of seven had the same pattern seen 18 h previously. Observations at three other sites were similar. Reappearance of the same strain(s) of SCN following sterilization of the site suggests that the reservoir for normal resident skin flora is located below the stratum corneum, perhaps in hair follicles and ducts of sebaceous glands.