An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (2.4 U/ml) and in deoxyribonuclease (0.02%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbecco's modified Eagle's medium and Ham's F 12 medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO2-incubator at 37 degrees C. After 2-3 wk cell outgrowths resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.