Everyone who has seen the flash of a firefly has observed the phenomenon of bioluminescence. Biological light production in various animals, plants, and bacteria has only recently been understood in molecular terms. Light production is dependent upon ATP in firefly tails (1) and reduced coenzymes such as NADH or FMNH2 in some bacteria (2). These findings form the basis for using bioluminescence as a sensitive analytical tool for detecting extremely small quantities of ATP and NADH down to 10"15 and 10"14 moles, respectively (3,4). Bioluminescent methods have wide-ranging utility for analysis; they have been used in detection of bacterial contamination, in determination of cell viability, in developing sensitive immunochemical assays, and in research areas where sensitive enzyme assays are required (3,5).Described below is a bioluminescent assay for ATP and NADH that meets the needs of an undergraduate biochemistry laboratory course. This experiment provides students with experience in both bioluminescence and analytical biochemistry yet requires limited instrumentation, preparation, and expense. The experiment as described can be run in one 3-hr time period and has potential for being used in conjunction with the measurement of metabolic levels of nucleotides and coenzymes.