The large (L) proteins of non-segmented negative stranded (NNS) RNA viruses contain the core RNA dependent RNA polymerase activity for RNA replication and transcription as well as the activities for polyadenylating and capping the mRNA transcripts and for methylating the cap structures. There is currently no structural information available for these large multi-functional proteins. Phylogenetic analyses have led to the division of the L protein primary structure into six functional domains of high conservation that are linked by variable regions. The studies in this report investigate the role of specific amino acids within domain VI of the VSV L protein, which contains a 2'-O-ribose methyltransferase (MTase) domain. We generated a structural homology model of residues 1644-1842 within domain VI based on the crystal structure determined for the known 2'-O-ribose MTase of E. coli, RrmJ. The information generated by this homology model directed us to residues structurally important for MTase activity and SAM binding. Selected residues were analyzed by site-specific mutagenesis and the mutant L proteins were assayed for their effects on RNA synthesis and cap methylation. The goal of this study was to functionally test the model in order to gain insight into the structural constraints of this region of the L protein. The data presented here revealed specific mutations that affect transcription, replication, and 5' cap methylation, many of which resulted in polymerases temperature sensitive for RNA synthesis.