The distance between specific sites on bacterial luciferase was estimated by energy transfer. Luciferase was fluorescently labeled by reaction of an essential sulfhydryl group with N-(l-pyrene)maleimide and N-[p-(2-benzoxa-zolyl)phenyl]maleimide. Both of the modified enzymes bind 8-anilino-l-naphthalenesuifonate (Ans) with affinities similar to that exhibited by the native luciferase. Using each of the two fluorescent probes as a donor and the bound Ans as an acceptor, the energy transfer efficiencies were determined by the resulting enhancement of fluorescence of the acceptor. The corresponding distance was calculated to be in the range of 21 to 37 A. Energy-transfer studies were also carried out using fluorescence lifetime measurements of bound ANS, acting as a donor with bound FMN as an acceptor. The corresponding distance was calculated to be between 30 and 58 Å. Using samples of luciferase:Ans complex and luciferase modified with N-(l-pyrene)maleimide, the rotational correlation time of the enzyme-dye conjugate as a whole was found to be 47 ± 2 ns. The observed rotational correlation time is much longer than that calculated for luciferase assuming a spherical structure, thus indicating an elongated form for the luciferase-dye conjugate.