The effect of the promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied on mixed primary cultures of C57BL/Icrf-at mouse submandibular gland and on slowly proliferating preneoplastic epithelial foci derived from such cultures. No cytologic alterations were noted in the TPA-treated epithelium. The development of three-dimensional epithelial ducts occurring between 30 and 60 days in vitro in untreated cultures was markedly inhibited in TPA-treated cultures. Epithelial proliferation, as shown by the [H]thymidine labelling index and mitotic index, seemded to increase during treatment with TPA and fall between the weekly treatments. Long-term TPA treatment increased the incidence of slow-growing foci in cultures previously treated with 7,12-dimethylbenz[a]anthracene but not in cultures treated with dimethyl sulfoxide. TPA had no effect on the emergence of tumorigenic cell lines at a late stage in culture or on the development of such lines from preexisting foci derived from non-TPA-treated cultures.