The glyUsuAGA mutation affects Escherichia coli tRNA Gl y GGG, changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base subsitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occurring spontaneously has been made also. The frequency of MNNG-induced glyUsuAGA mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyAGA mutations, however, strongly suggests that the occurrence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position. In addition to the double change in the anticodon, the glyUsuAGA tRNA differs from tRNA Gl y GGG in that it bears a modification of the A adjacent to the 3' position of the anticodon. Most likely, this modified base is N-[9-(beta-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine.