Background Long‐term cultures of epidermal cells from mouse skin have been notoriously difficult to establish.

Objectives To develop a modified serum‐free medium and technique for long‐term culture of adult mouse epidermal keratinocytes.

Methods Epidermal cells from trypsin‐treated adult mouse dorsal and ventral skin were grown on type I collagen‐coated dishes without feeder layers in a serum‐free medium supplemented with only 10 ng mL⁻¹ epidermal growth factor (EGF) and 10⁻¹⁰ mol L⁻¹ cholera toxin (CT).

Results After removing coexisting fibroblasts several times, we were able to obtain almost pure basal epidermal keratinocytes. Our technique supports sustained multiplication of mouse basal keratinocytes for more than 100 population doublings, and they retained the capacity to undergo terminal differentiation when given the appropriate stimulus. The epithelial nature of these cultivated cells was demonstrated both by phase‐contrast microscopy and by immunostaining with antikeratin antibodies. EGF and CT, which have been reported to accelerate the cellular growth rate, were essential for successful long‐term cultivation during multiple passages.

Conclusions Our technique is very simple. It provides a useful and suitable model for investigations of growth, differentiation and skin remodelling in vitro.