We have shown the increase in the acetyl-CoA-independent activity of sheep liver pyruvate carboxylase following trinitrophenylation of a specific lysine residue (designated Lys-A) to be the result of a large stimulation of the first partial reaction and a slight stimulation of the second partial reaction catalysed by this enzyme. Like acetyl-CoA, the activators adenosine 3',5'-bisphosphate and CoA did not stimulate the catalytic activity of the trinitrophenylated enzyme in either the overall reaction or the first partial reaction. Conversely, trinitrophenylation had no effect on activation of the overall reaction and the second partial reaction by acetyl-phosphopantetheine. Protection experiments demonstrated that the presence of both acetyl-CoA and adenosine 3',5'-bisphosphate decreased the rate of loss of activity during exposure of sheep liver pyruvate carboxylase to trinitrobenzenesulphonic acid (TNBS), whereas acetyl-phosphopantetheine did not. 5'-AMP and acetyl-dephospho-CoA did not protect the enzyme against loss of activity, whereas the presence of adenosine 2',5'-bisphosphate only slightly decreased the rate of modification. This suggests that Lys-A interacts with the adenosine nucleotide portion of the acetyl-CoA molecule, specifically the 3'-phosphate moiety. Acetyl-CoA and adenosine 3',5'-bisphosphate were shown to protect pyruvate carboxylase from Saccharomyces cerevisiae against inhibition by TNBS. A [14C]acetyl-CoA-binding assay demonstrated that modification of Lys-A inhibits the binding of acetyl-CoA to S. cerevisiae pyruvate carboxylase, indicating that Lys-A is at or near the acetyl-CoA-binding site.