Increasing cell size, lipid accumulation, and altered antigen expression are features of sebaceous differentiation in vivo. Enhanced lipid synthesis with progressive differentiation is also present in cultured human sebocytes. This study was conducted to investigate the evolution of cell size and antigen expression of human sebocytes with progressive differentiation in vitro. Subconfluent human sebocyte cultures were examined for sebocyte differentiation evaluated on cytocentrifuge preparations by light microscopy and classified in stages according to morphological criteria described for sebocytes in vivo. Rates of 5.1 +/- 2.2% undifferentiated sebocytes, 29.2 +/- 4.9% early differentiated, 20.7 +/- 4.1% advanced differentiated, 37.6 +/- 6.4% fully differentiated, and 5.9 +/- 1.9% mature sebocytes were calculated in secondary cultures. The size of cultured sebocytes measured by computer-assisted planimetry significantly increased with progressive differentiation up to 4-5.5 times. The low rates of mature sebocytes and the only moderate increase of their size with progressive differentiation indicate an incomplete terminal differentiation in vitro. Sebocytes were subsequently stained with a series of monoclonal antibodies (mAb) to determine antigen expression using the alkaline phosphatase anti-alkaline phosphatase technique. The number of sebocytes labeled with the anti-keratin mAb CK8.12 and KL1, and the mAb 34D11 (82 kD protein) increased with progressive differentiation; significant differences were found after comparing early and advanced differentiated sebocytes. Sebocytes were positively stained with the anti-keratin mAb 6B10 (K 4), RPN1162 (K 7), CK13 (K 13), RPN1165 (K 19), CK8.60, and the mAb 115F5 (MAM-6c), OM-1 (sebaceous gland antigen), and 24F10 (basic polypeptides) only at late-stage differentiation. The expression of keratins 4, 7, 13, and 19 was confirmed by gel electrophoresis and immunoblotting. The data obtained were used to study the effects of the duration of cultivation and of the retinoids isotretinoin and tretinoin on sebocyte differentiation in vitro. Subcultivation of sebocytes upregulated, and treatment with isotretinoin but not with tretinoin downregulated labeling with mAb which recognize indicating progressive and late-stage differentiation.