Abstract The structures of e-41 dinitrophenyl-ribonuclease-S (e-41-DNP-RNase-S) and its complexes with 3'-pyrimidine nucleotides have been determined crystallographically at 2 and 3 A resolution, respectively, using the difference Fourier technique. In addition, the binding of Cu2+, UO22+, and PtCl4-2 to crystalline RNase-S and e-41-DNP-RNase-S have been compared. 1. The 2,4-dinitrophenyl (DNP) group of crystalline e-41-DNP-RNase-S binds to the protein in a cleft bounded by the side chains of Gln-11, Asn-34, Asp-38, and Arg-39, and the protein backbone between Asn-34 and Asp-38. It does not physically block the active site. As a result of the interaction between the DNP group and the enzyme, the distance between the e-amino group of Lys-41 and the phosphate group bound at the active site increases by 3 A. 2. There are conformational differences between RNase-S and e-41-DNP-RNase-S which require protein shifts of at least 0.5 to 1.0 A throughout the entire molecule. Some of these shifts may result from the effect of the DNP groups upon the crystal packing. 3. 3'-Pyrimidine nucleotides bind to crystalline RNase-S and e-41-DNP-RNase-S in the same way, and with no significant difference in their binding constants. 4. The binding of 3'-pyrimidine nucleotides to crystalline e-41-DNP-RNase-S reduces the magnitude of the conformational difference between it and RNase-S. However, the position of the DNP group is not affected. 5. The positions or occupancies of several metal ion-binding sites are slightly different in crystalline RNase-S and e-41-DNP-RNase-S.