The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro. RNA tumor virus particles such as murine leukemia virus or Rous sarcoma virus (RSV) contain two major size classes of RNA, the high-molecular-weight genome RNA and lowmolecular-weight 4 to 7S RNA (1-3, 7, 8, 12, 19, 21, 25, 27). Some of the low-molecular-weight RNAs are associated with the genome RNA in a 70S complex, whereas others are released free in solution after disruption of virus particles (8, 12, 19, 21, 27). One of the 70S-associated 4S RNAs in RSV virions, the host cell tRNA'r , has been shown to be the primer for initiation of RSV RNA-directed DNA synthesis in vitro (6, 11, 14, 22). To determine whether other viruses use tRNA's as primers, we analyzed the small RNAs of Moloney murine leukemia virus (M-MuLV). One ofthe 70S-associated 4S molecules can serve as the major primer for reverse transcription in vitro. This RNA dissociates from the genome RNA at higher temperatures than do other associated species. An identical molecule is found in uninfected mouse, rat, and chicken cells, suggesting that the M-MuLV primer is a host cell RNA. In a later communication, we will show that this molecule has the amino acid acceptor activity and nucleotide sequence of tRNApro. MATERIALS AND METHODS Virus growth and isolation. The cloned line of M-MuLV designated clone 1 was propagated in a 3T3