When treated with phorbol tumor promoters, HL-60 cells undergo terminal differentiation evidenced by a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity. Internalization of fluorescent particles by cells exhibiting the phagocytic positive phenotype (phag+) provides a sensitive indication of promoter-induced differentiation, and the resulting fluorescent cells can be quantitatively analyzed by flow cytometry. The current study was initiated to further test the predictive power of a flow cytometry based HL-60 differentiation assay in the detection of agents associated with tumor promotion. Specifically, experiments were designed to assess the sensitivity of the test system to co-promoters which enhance promoter activity in vivo. Prostaglandin E2 (PGE2) was chosen as a model co-promoter since it has been shown to potentiate phorbol ester (i.e. 12-O-tetradecanoyl phorbol-13-acetate; TPA) induced biological effects in vivo. Results detailed in the current report indicate that PGE2 enhances TPA-induced differentiation of HL-60 cells in a dose-dependent manner. As with in vivo co-promotion experiments, PGE2 exhibited a maximum potentiating effect when administered prior to TPA. These data indicate that HL-60 cells are not only sensitive to phorbol promoters, but also to the co-promoter PGE2. These experiments support the hypothesis that a flow cytometry based HL-60 assay may prove useful for studying chemical agents or intrinsic cellular factors that are involved in the tumor promotion phase of carcinogenesis.