Abstract: Previous animal studies have demonstrated that α-melanocyte-stimulating hormone (α-MSH) is a sebotropic hormone in rats and that targeted disruption of melanocortin 5 receptor (MC5-R) can down-regulate sebum output in mice. To study the role of proopiomelanocortin (POMC) peptides in the regulation of human sebaceous lipid production and sebocyte differentiation, we established a primary human sebocyte culture system. Sebocytes were derived from normal human facial skin. Differentiation of sebocytes, induced by POMC-derived peptides such as MSH, adrenocorticotropic hormone (ACTH), or bovine pituitary extract (BPE), resulted in the appearance of prominent cytoplasmic lipid droplets. Partial induction of sebocyte differentiation also was observed in serum-depleted cultures, but there was very limited spontaneous differentiation in serum-containing medium. Analysis by high-performance thin-layer chromatography (HPTLC) of 14C-acetate-labeled lipids showed a dose-dependent increase in synthesis of sebaceous-specific lipid (i.e., squalene) induced by NDP α-MSH. Molecular studies using RT-PCR showed a low level of human MC5-R expression under serum-free condition but a substantial increase after treatment with NDP α-MSH or BPE. In contrast, MC1-R expression remained the same, independent of treatment. Our data indicate that expression of MC5-R correlates with sebocyte differentiation and suggest a regulatory role for MC5-R in human sebaceous lipid production.