The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.