The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F420 and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F420 fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F420 from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.