Studies were carried out to examine the susceptibility of normal and initiated tracheal epithelial cells of rats to known tumour-promoting agents. The ability of normal rat tracheal epithelial cells to form colonies in cultures was enhanced markedly by addition of known tumour promoters to the medium. Several retinoids were shown to reduce the ability of these cells to form colonies in culture and to inhibit this effect of 12-O-tetradecanoylphorbol-13-acetate (TPA). A transformation assay of tracheal epithelial cells was used to study promotional effects of TPA in cultures initiated with N-methyl-N'-nitro-N-nitrosoguanidine. In this assay, four stages of transformation are recognized. TPA did not affect measurably the first two transformation stages, i.e., the development of transformed colonies (enhanced growth variants) and the 'immortalization' of enhanced growth variant-derived subcultures. However, treatment of cultures with TPA during the early post-initiation period resulted in a marked enhancement of the appearance of the third transformed phenotype, which is characterized by anchorage independence of growth. The findings of these in-vitro initiation-promotion studies paralleled, in all major respects, the results obtained in in-vivo - in-vitro studies. Tracheas exposed in vivo to initiator and promoter were shown to develop the same transformants observed in the in-vitro assay. TPA affected primarily the late anchorage-independence phenotype. Parallel tumour induction studies showed that TPA increased markedly the incidence of tracheal carcinomas following initiation with a low dose of 7,12-dimethylbenz[a]anthracene. The studies thus demonstrate that TPA is an effective tumour promoter for rat tracheal epithelium, causing an increase in tracheal carcinomas. They further suggest that the action of TPA on an early transformed cellular phenotype enhances the development of later phenotypes, including neoplastic cell variants.