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In this study, we wanted to see if fluorescence anisotropy could be used to detect changes in metabolism in cells with significant light scattering and absorption properties. Fluorescence anisotropy measurements of nicotinamide adenine dinucleotide (NADH) were performed with human melanocytes and melanoma cell lines. To demonstrate the feasibility of using fluorescence anisotropy for detecting metabolic changes, the electron transport chain was blocked using rotenone, inducing an accumulation of intracellular NADH. Total fluorescence increased in all cells as a result of rotenone treatment. Fluorescence anisotropy decreased in the rotenone-treated cells relative to the controls, suggesting an increased ratio of free to protein-bound NADH in the treated cells. In general, the fluorescence anisotropy of the melanocytes was significantly higher than that of the melanoma cell lines. Reflectance spectroscopy showed that the differences in fluorescence anisotropy between the cell types were not due to differences in scattering and absorption properties. Intrinsic cellular NADH fluorescence was experimentally extracted by ratioing polarized fluorescence to polarized reflectance. NADH binding, measured as the ratio of fluorescence intensity at 430 and 465 nm, showed more protein-bound NADH in the melanocytes than in the melanoma cells, consistent with the fluorescence anisotropy measurements.