Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP(i). The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PP(i) strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 μM, 260 μM and 42 μM for ATP, malonate and CoA respectively, and the value for V(max) was 11.2 μM/min. The t.i.c. analysis of the 32P-labelled products in a reaction mixture containing [γ-32P]ATP in the absence of CoA showed that PP(i) was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using ([)(18)O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.i.c. analysis of reaction mixture containing [α-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Ping Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.