The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release [33P]UMP when incubated with a RNA.DNA hybrid containing a [33P]UMP-labeled RNA strand. The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator. RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent. A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion. The radiolabeled RNA.DNA hybrid or DNA.DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times. The DNA.DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration. However, nearly complete digestion of the RNA.DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA.DNA hybrid vs the DNA.DNA duplex. Time course reactions of the enzymatic digestion of the 3'-labeled RNA.DNA hybrid or DNA.DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.