MMLV reverse transcriptase (or M-MuLV reverse transcriptase) is coded by Moloney murine leukemia virus. MMLV reverse transcriptase is an RNA-dependent DNA polymerase that synthesizes the first complementary DNA strand (cDNA) from a single-stranded RNA model to which a primer has been hybridized. This enzyme is also capable of extending the hybridized primers to single-stranded DNA. The synthesis of the second strand of the cDNA can be obtained from certain mRNA models without additional DNA polymerase. M-MLV reverse transcriptase is used for the preparation of cDNA libraries or for cDNA synthesis by Reverse-Transcription PCR (RT-PCR).

M-MuLV reverse transcriptase lacks 3 '→ 5' exonuclease activity. Compared to AMV reverse transcriptase, MMLV has no endonuclease activity and has a lower RNase H activity. RNase H (Ribonuclease H) is an endoribonuclease that specifically hydrolyses the phosphodiester bonds of RNA that is hybridized to DNA. This enzyme does not digest single-stranded or double-stranded DNA.