As a first step toward the development of stable, selective, and potent inhibitors of those members of the pancreatic RNase superfamily that induce biological responses, we have focussed on low molecular weight compounds and studied their interactions with the active-site of bovine pancreatic ribonuclease A (RNase A). A new inhibitor is described, 5'-diphosphoadenosine 3'-phosphate, which binds to RNase A more tightly than any previous low molecular weight compound: its Ki value of 1.3 microM at pH 7 is 8-fold lower than that for uridine-vanadate, a transition-state analog, and 110-fold lower than that for 2'-CMP, one of the best-characterized RNase A ligands. The new inhibitor is found to contact RNase A residues that are conserved in several homologous mammalian RNases and hence should be able to serve as a basis for the design of even tighter-binding inhibitors of these enzymes.