The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.