L cell DNA ligase catalyzes a covalent linkage between 5'-hosphoryl oligodeoxyribonucleotides and 3'-hydroxyl oligoribonucleotides on a complementary polydeoxyribonucleotide template. This reaction occurs to a substantially lesser extent than does the sealing of DNA to DNA. The joining of [5'32P]d(pA)12-18 to (Ap)11A on poly[d(T)] or of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) ON POLY[D(C)] was demonstrated by the formation of alkaline phosphatase resistant radioactivity. The 32P of the hybrid reaction products became sensitive to the action of alkaline phosphatase after treatment with alkali. Furthermore, hydrolysis of the products of the linkage of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) on poly[d(C)] with micrococcal nuclease and spleen phosphodiesterase resulted in the formation of [3'-32P]IMP. Attempts to seal [5'-32p[-(pA)12 to d(Ap)11-17A on poly[d(T) or [5'-32P]oligo(pI) to d(Gp)11-17G on poly[d(C)] were unsuccessful.