We explored the mechanism of the stabilization of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) by eliminating RNase H activity. Without the template-primer (T/P) poly(rA)-p(dT)(15), the temperature reducing initial reverse-transcription activity by 50% over a 10-min incubation of the RNase H activity-deficient variant D524A was higher by 3.7 degrees C than that of the wild-type enzyme (WT). In the reverse transcription reaction, the K-m values for T/P of WT and D524A were almost the same. These results suggest that elimination of RNase H activity enhanced the intrinsic thermal stability of MMLV RT rather than its affinity toward T/P.