Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M(r)) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M(r) values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (alpha : 45,000, beta : 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5-8.0 and 30-35-degrees-C, respectively. The citrate synthase was stable in the pH range of 6.0-9.0 and up to 55-degrees-C. The apparent K(m) values for oxaloacetate and acetyl-CoA were about 27 muM and 410 muM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.