The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on survival, proliferation, and differentiation of normal and preneoplastic rat tracheal epithelial (RTE) cells. Normal RTE cells obtained from 8-week-old rats were plated into medium containing TPA at concentrations ranging from 0.1 to 100 ng/ml. A dose-related increase in colony-forming efficiency (CFE) was observed at concentrations above 0.1 ng/ml. At 100 ng/ml the CFE was increased 5-fold above that observed in controls. Similar studies were carried out with immortal, preneoplastic RTE cells derived from carcinogen-exposed primary cultures. Upon exposure to 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decrease in CFE ranging from a 70 to a greater than 99% reduction. Two out of 11 cell lines showed only a minor reduction in CFE at this TPA concentration. Teleocidin B was as potent as TPA in causing inhibition of CFE in preneoplastic RTE cells; the potency of 4-O-methyl TPA was approximately 1/100 that of TPA and phorbol was virtually inactive. Further studies suggested that inhibition of clonal growth of the preneoplastic tracheal cells by TPA was due to rapid cell death. As early as 2 h after addition of TPA to the cultures, a loss of viable cells from the cultures as well as a reduction in CFE was observed. Morphological studies support the interpretation that a large number of cells was severely damaged within 2--4 h after TPA exposure. The formation of cross-linked envelopes (CLE), a measure of keratinocyte differentiation, was determined in cultures of preneoplastic RTE cells. TPA caused only minor increases in CLE formation indicating that the cells were not dying from TPA-induced terminal keratinocyte differentiation, but rather from TPA toxicity. Thus, preneoplastic RTE cells respond differently to TPA from normal RTE cells and also from normal and transformed keratinocytes of various origins. The role of TPA toxicity in promoting preneoplastic RTE cells to the neoplastic state is being investigated.