In order to make efficient use of T4 RNA ligase in a program involving chemical-enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High-performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5')pp(5')Cp or A(5')pp(5')UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Conversely, examination of the four nucleoside 3',5'-bisphosphate donors pUp, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.