Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized. Using Eagle's medium with 0.05 mM Ca 2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas. When tested for tumorigenicity in nude mice by injection of a cell suspension or implanation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage. In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested. The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative. When tested for responsiveness to Ca 2+ (>0.1 mM) as a differentiation stimulus, all lines responded. In the higher Ca 2+ medium there was a 50–95% decrease in colony-forming efficiency, a slight decrease in [ 3 H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF). Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca 2+ >0.1 mM. However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca 2+ while the remaining cells continued to proliferate, although at a slower rate. Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines. 12- O -Tetradecanoyl-phorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 0.05 mM Ca 2+ or 1.2 mM Ca 2+ . TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca 2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca 2+ . TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes. Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation. All of these properties should provide a growth advantage to these cells during tumor promotion.