- The substrate specificity of T4 RNA ligase has been examined to determine whether the intermolecular reaction is sufficiently general to realize its potential in the enzymatic synthesis of oligoribonucleotides of defined sequence. Reactions between a variety of acceptor molecules with 3'- and 5'-bydroxyl groups and donor molecules with 3'- and 5'-phosphates indicate that the minimal substrates are a trinucleoside diphosphate acceptor and a nucleoside 3',5'-bisphosphate donor. Increasing the chain length of either the acceptor or donnor has little effect on the rate or extent of reaction. Although the base composition of the donor has only a small effect on the reaction rate, the presence of uridine in the acceptor greatly reduces the amount of product formed. The presence of a phosphate on the 3' terminus of the donor molecule permits a unique intermolecular product with a 5'-hydroxyl and a 3'-phosphate. By enzymatically either adding a 5'-phosphate or removing the 3'-phosphate, a new donor or acceptor is prepared so synthesis of an oligomer chain can proceed in either direction. With the simplicity of this enzymatic pathway and the rather broad substrate specificity of T4 RNA ligase, a convenient method for the synthesis of oligoribonucleotides is established.