We have examined colony-forming ability, localization of colony-forming cells, and in vitro life spans of outer root sheath keratinocytes of different fragments of adult human plucked hair follicles. These were shown by immunohistochemical staining for cytokeratins and integrins to contain a preserved basal cell layer. By microdissection, five fragments of the outer root sheath (B1, B2, B3-1, B3-2, B4) were separated, dispersed by trypsin into single cell suspensions, and grown on human feeder fibroblasts. All fragments gave rise to at least some colonies, but colony-forming ability was mostly marked in the intermediate part (B2) and the lower half of the central part (B3-1); approximately 60% of colony-forming cells of a hair follicle localized to the fragment B3-1 and 28% to the fragment B3-2 (upper half of the central part, including bulge). To compare the in vitro life spans of cells from the various fragments, we subcultured isolated keratinocytes under identical conditions. The longest was found in the fragment B3-2 and the shortest in the fragment B1 (bulb). Moreover, the differentiation state of the native cells and the cells of all cultures were studied during their whole life spans by immunocytochemical analysis of various proliferation and differentiation markers. Surprisingly, keratinocytes of all fragments, as shown by expression of high-molecular-weight cytokeratins and filaggrin, were capable of terminal differentiation. These data indicate that cells with long life spans are localized in central parts of the outer root sheath close to the bulge area and that cells with high colony-forming ability are localized in the lower central parts. The latter are usually removed by plucking and may therefore not represent stem cells but rather cells important for hair growth during a single cycle. Cells with long life spans--also included in plucked hair follicles--may be immediate progeny of stem cells that will be segregated in the bulge area. Finally, our results are important for gene transfer and stem cell gene therapy in genodermatoses, because plucked hair follicles are easily available and keratinocytes close to the bulge area should be used selectively.