The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.