The BALB/c-3T3 cell neoplastic transformation system was modified to examine the tumor promoting activity of a set of substances. Following initiation of the target cells with 3-methylcholanthrene, treatment of the cultures with phorbol myristate acetate (0.01 microgram/ml; 1.5 X 10(-8) M) during the remainder of the 4-week assay interval resulted in a marked increase in both spontaneous and initiated Type III transformed foci. In contrast, a similar treatment with saccharin at 20, 100 or 500 microgram/ml (0.08, 0.4 or 2.1 X 10(-3) M) did not influence the occurrence of Type III transformed foci and did not result in a promoting response. Sodium ascorbate (2.53 X 10(-3) M) and L-tryptophan (2.45 X 10(-3) M) almost completely inhibited both spontaneous and initiated Type III transformed foci. Calcium pantothenate (2.10 X 10(-3) M) exhibited a marginal promoting effect. Under the conditions of this study in which the classical tumor promoter phorbol myristate acetate was highly active in promoting Type III transformed foci, saccharin was not active as either a direct transforming or promoting agent at doses up to 5 orders of magnitude higher.