The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NAD(P)H fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior-probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the time range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component as most pronounced for the intact strain DL1. Time-resolved fluorescence seems therefore to be an appropriate method to probe the function of the respiratory chain and-in the further step-to differentiate between various types of cells and tissues in medical diagnosis or environmental research.