tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA(His) guanylyltransferase accumulate uncharged tRNA(His) lacking the G(-1) residue and subsequently accumulate additional 5-methylcytidine (m(5)C) at residues C(48) and C(50) of tRNA(His), due to the activity of the m(5)C-methyltransferase Trm4. We show here that the increase in tRNA(His) m(5)C levels does not require loss of Thg1, loss of G(-1) of tRNA(His), or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA(His) m(5)C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m(5)C in tRNA(His) after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m(5)C on tRNA(His) occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m(5)C appears specific to tRNA(His), as tRNA(Val(AAC)) and tRNA(Gly(GCC)) have much reduced additional m(5)C during these growth arrest conditions, although they also have C(48) and C(50) and are capable of having increased m(5)C levels. Thus, tRNA(His) m(5)C levels are unusually responsive to yeast growth conditions, although the significance of this additional m(5)C remains unclear.