In an x-ray diffraction study by the isomorphous replacement method, the structure of the complex of aspartate carbamoyltransferase (EC 2.1.3.2) bound to the bisubstrate analogue N-(phosphonacetyl)-L-aspartate has been solved to 2.9-A resolution (R = 0.24). The large quaternary structural changes previously deduced by molecular replacement methods have been confirmed: the two catalytic trimers (c3) move apart by 12 A and mutually reorient by 10 degrees, and the regulatory dimers (r2) reorient each about its twofold axis by about 15 degrees. In this, the T-to-R transition, new polar interactions develop between equatorial domains of c chains and the Zn domain of r chains. Within the c chain the two domains, one binding the phosphonate moiety (polar) and the other binding the aspartate moiety (equatorial) of the inhibitor N-(phosphonacetyl)-L-aspartate, move closer together. The Lys-84 loop makes a large relocation so that this residue and Ser-80 bind to the inhibitor of an adjacent catalytic chain within c3. A very large change in tertiary structure brings the 230-245 loop nearer the active site, allowing Arg-229 and Gln-231 to bind to the inhibitor. His-134 is close to the carbonyl group of the inhibitor, and Ser-52 is adjacent to its phosphonate group. However, no evidence exists in the literature for phosphorylation of serine in the mechanism. Residues studied by other methods, including Cys-47, Tyr-165, Lys-232, and Tyr-240, are too far from the inhibitor to have a direct interaction.