The functional role of essential residue α-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants αR376C, αR376Q, and αR376K were constructed, and combined with the mutation βY331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of α-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that α-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the α-Arg-376 guanidinium group exists, likely in α/β conformational signal transmission required for steady-state catalysis. Parallels between α-Arg-376 and GAP/G-protein 'arginine finger' residues are evident.