We present an improved method for the isolation and cultivation of human scalp anagen hair follicle dermal papilla cells. Following treatment of the isolated dermal papilla with collagenase, incubation in Chang's medium mediates accelerated growth of the papilla cells when compared with other media such as DMEM, M199, and EMEM. Upon reaching confluency, the cells cultured in this fashion exhibit a multilayer-forming property that is dependent on normal proteoglycan synthesis. The papilla cells maintain this morphologic behavior for as long as 7 weeks in culture, or after being subcultured six times. During this time, the cells continue to synthesize extracellular matrix components associated with the human anagen follicle in situ. These include chondroitin sulfate, laminin, and type IV collagen. Type III collagen and keratan sulfate are poorly expressed by the papilla both in situ and in vitro. Heparan sulfate proteoglycan, a matrix component of the papilla in situ, is poorly expressed in vitro. Earlier reports suggested that the expression of extracellular matrix components is not maintained in culture. We show that the expression of these molecules is not dependent on the secondary culture medium, but continues in DMEM and M199 after primary culture in Chang's medium. Our results suggest that initial exposure of the dermal papilla to Chang's medium either selectively permits the outgrowth of papilla cells having extracellular matrix components similar to those found in situ, or stabilizes the expression of extracellular matrix components among the entire cultured cell population.