With the use of ultraviolet absorbance at around 335 mμ, which is specific for 4-thiouracil residue in native Escherichia coli tRNA, the behavior of the 4-thiouridine region in response to heat denaturation has been investigated in comparison with that of the whole region by using seven individual amino acid-specific tRNA's. The data indicated that the melting out temperature of the 4-thiouridine region depended upon the amino acid-specific tRNA species used. In addition, the 4-thiouridine region in many of the tRNA species melted out in bi-modal fashion. Further study of the outstanding bi-modal melting of the 4-thiouridine region in tRNAMetF has indicated that a local rearrangement in the tRNA molecule occurred at around 70°, which was between the two phases. The discrepancy between the bi-modal behavior of 4-thiouridine and the fact that only one 4-thiouridine residue per tRNA molecule is present could be interpreted to suggest that the two-dimensional structure of tRNA was further folded to construct a more complicated form. © 1969.