The Escherichia coli tyrosine amber suppressor tRNA gene, supF, has been utilized as a mutagenic target in several shuttle-vector plasmids. Data on mutagenic inactivation of suppressor activity was obtained from induced mutagenesis experiments with plasmids pZ189 and p3AC, and from studies on alterations of the supF gene transduced into E. coli. 162 single or tandem base-substitution mutations that reduce or eliminate suppressor activity were identified at 86 sites within 158 base pairs. The 2 transition and 4 transversion mutations possible in double-stranded DNA were all detectable. At 56 sites two different inactivating mutations were found; and at 20 sites all 3 possible base substitution mutations inactivated suppressor function. Most of the mutations were clustered within the mature tRNA region: 144 of the base-substitution mutations were found at 74 sites within the 85-bp mature tRNA region. Insertions of 1 or 2 bases at 4 sites and deletions of 1 to 3 bases at 15 sites were found to inactivate supF function. A few silent mutations which do not inactivate suppressor function were found: single base-substitutions at 4 sites, 14 pairs of silent double mutations, and a large deletion including the promoter region. The supF gene is thus an extremely sensitive target for mutagenic inactivation in shuttle-vector plasmids.