A novel RNA ligase activity has been detected in a cell-free extract from Rhodosporidium toruloides. The enzyme system was found to successively join 2`-, 3`-, or cyclic 2` : 3`-nucleotide to the 5`-phosphate of synthetic U_(16)G, leading to the synthesis of N_nU_(16)G(n=1,2,3...). The ligation of the nucleotides with synthetic oligonucleotide was found to proceed by forming 2`-phosphomonoester, 3`, 5`-phosphodiester intermediate linkage. The RNA ligase requires ATP and Mg^(2+) for its maximum activity. Interestingly, treatment of the cell- free extract with micrococcal nuclease but not with deoxyribonuclease abolishes the enzyme activity, indicating that the RNA ligase requires an RNA component(s) for its ligation activity.