The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), induces macrophage characteristics in the HL-60 promyelocytic leukemia cell line. These same cells can be induced to develop mature myeloid characteristics with a variety of other stimuli. Since normal colony-forming units-culture (CFU C) also have the dual capability of developing colonies with myeloid or monocyte characteristics, the effect of TPA on normal human CFU-C development was studied. To carry out these studies, a method was developed to identify cells histochemically within agar cultures as containing either the myeloid marker, chloroacetate esterase (CAE), or the monocyte marker, nonspecific esterase (NSE). Cells from normal donors were placed into agar cultures with placenta conditioned medium (PCM), TPA in various concentrations, or combinations of PCM and TPA as stimulating materials, and examined after 7-14 days of incubation. TPA alone at concentrations of 5 x 10(-7) M to 10(-9) M stimulated cluster formation. With increasing concentrations of TPA, the percentage of clusters positive for NSE progressively increased, while CAE-positive clusters decreased. I contrast, when TPA at concentrations greater than 10(-9) M was added to PCM, the total number of clusters and colonies decreased. This resulted from a decrease in the number of clusters and colonies that contained the myeloid marker CAE, whereas the number positive for the monocyte marker NSE remained unchanged. These studies demonstrate two effects of TPA on normal CFU-C. In the absence of other sources of colony stimulating activity (CSA), TPA induces clusters. In the presence of PCM, it inhibits the production of myeloid colonies and clusters. Under both conditions, it favors the development of colonies and/or clusters containing predominantly monocytes.