Several coumarin-labeled nucleotides have been synthesized, based on 2'(3')-O-(2-aminoethyl)carbamoyl-ATP (edaATP). The fluorescent coumarins coupled with the free amino group are 7-diethylaminocoumarin-3-carboxylic acid (to give deac-edaATP), coumarin 343 (but-edaATP) and 7-ethylamino-8-bromocoumarin-3-carboxylic acid (mbc-edaATP). The carbamoyl linkage of these nucleotide analogs undergoes interconversion between 2'- and 3'-hydroxyl attachment very slowly, so that the 2'- and 3'-isomers were separated and stored with minimal equilibration. 3'-Deac-edaADP had fluorescence excitation and emission maxima at 430 nm and 477 nm, with a fluorescence quantum yield of 0.012. The equivalent data for 3'-but-edaADP are 445 nm, 494 nm, and 0.51, respectively, and for 3'-mbc-edaADP, 405 nm, 464 nm, and 0.62. The interaction with skeletal myosin subfragment 1 was measured in the absence and presence of actin. In each case the fluorescence was decreased when bound to subfragment 1, 3-fold for 3'-deac-edaADP, 7-fold for 3'-but-edaADP, and 11-fold for 3'-mbc-edaADP. Steady-state ATPase measurements and the kinetics of binding and release of nucleotides were similar to those reported for the natural nucleotide. Large fluorescence changes could be observed for the release of these analogs from actomyosin subfragment 1, enabling a direct measurement of the kinetics of this process. In the case of 3'-deac-edaADP a rate constant of 474 s(-1) was measured (at pH 7.0, 20 degrees C, and low ionic strength).