The fluorescent dyes, rhodamine 6G and 123, which specifi-cally stain mitochondria, were used to examine changes in mitochondria that follow malignant transformation. The spatial distribution and shapes of mitochondria differ in untransformed and malignant-transformed cells. In untransformed C3H/10T1/2 clone 8 cells, the mitochondria were distributed radially around the nucleus, and each had a fibrous shape. In chemically transformed MCA clone 16 cells, the mitochondria were distributed randomly in the cytoplasm, and each was shaped like a short rod. Another important mitochondrial change after malignant transformation was the change in the time course of fluorescence emission from the rhodamine present in the mitochondria. A slow increase in fluorescence, which was instantaneous at the time of excitation irradiation occurred in untransformed but not in transformed cells. This slow fluorescence emission, peculiar to untransformed cells was affected by proton ionophore but not by calcium ionophore treatment. The difference in the time courses of fluorescence emission for untransformed and transformed cells may reflect differences in the quenching of the dye fluorescence. The data reported provide evidence that mitochondria are affected by malignant cell transformation.