Molecular dynamics simulations of the active site of RNase A have been used to supplement structural and chemical data. A simulation of the CpA substrate complex with a neutral His 12, based on the X-ray structure with deoxy-CpA and protonated His 12, demonstrates a repositioning of important residues that permits them to fulfill their catalytic role; i.e., His 12 is oriented with NE2 in place to accept the proton from the 02’ hydroxyl (instead of interacting with the phosphate oxygen, as in the X-ray structure), and Lys 41 is sufficiently close to aid in the proton transfer required for transphosphorylation (instead of being far from the substrate, as in the X-ray structure). Protonated His 119 interacts with a phosphate oxygen, in position to polarize the phosphorus or transfer a proton, as in the Breslow mechanism. Simulations are also reported for complexes with an intermediate (cyclic CMP) and a product (3’-UMP). An analysis is presented of the residues, other than His 12 and 119 and Lys 41, that may play a significant role in catalysis; they include Thr 45, Gin 11, Phe 120, Lys 66, and Asp 121. Lys 7 does not hydrogen bond to the substrate and appears not to participate directly in the reaction. © 1990, American Chemical Society. All rights reserved.