When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment to Ulex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin.(ABSTRACT TRUNCATED AT 250 WORDS)