The 3-methylcytidine (m 3 C) modification is ubiquitous in eukaryotic tRNA, widely found at C 32  in the anticodon loop of tRNA Thr , tRNA Ser , and some tRNA Arg  species, as well as in the variable loop (V-loop) of certain tRNA Ser  species. In the yeast  Saccharomyces cerevisiae , formation of m 3 C 32  requires Trm140 for six tRNA substrates, including three tRNA Thr  species and three tRNA Ser  species, whereas in  Schizosaccharomyces pombe , two Trm140 homologs are used, one for tRNA Thr  and one for tRNA Ser  The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how  S. cerevisiae  Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G 35 -U 36 -t 6 A 37  of the anticodon loop of tRNA Thr  substrates, and this sequence is an identity element because it can be used to direct m 3 C modification of tRNA Phe  However, Trm140 recognition of tRNA Ser  substrates is different, since their anticodons do not share G 35 -U 36  and do not have any nucleotides in common. Rather, specificity of Trm140 for tRNA Ser  is achieved by seryl-tRNA synthetase and the distinctive tRNA Ser  V-loop, as well as by t 6 A 37  and i 6 A 37  We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates m 3 C modification of tRNA Ser(CGA)  and tRNA Ser(UGA)  in vitro. In addition, our results show that Trm140 binding is a significant driving force for tRNA modification and suggest separate contributions from each recognition element for the modification.