Continuously growing cell cultures, testing positive for tyrosinase activity, were derived from two brain and three lymph-node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation fate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 f 130 p M BrdUrd (range: 130-520; n = 11) vs. 650 f 50 pM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhi- bition at 200 2 55 pM (range: 65-400 FM) for 20% oxygen vs. 360 f 130 p M BrdUrd for 5% oxygen. The cell cycle kinetic mechanism of BrdUrd-induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3-fold variation in BrdUrd sensitivity. I n one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 3325Wethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy.