Resistance to streptomycin (Sm) of Euglena gracilis chloroplasts can be due to a single C to T transition of the 16S rRNA gene in an invariant position which is equivalent to C912 of the Escherichia coli 16S rRNA. Since Euglena chloroplasts cannot be transformed we introduced, by site-directed mutagenesis, a C912 to T transition in the cloned rrnB operon (pKK3535) of E. coli and used this new construct (pEM109) in transformation experiments. Transformed E. coli cells were selected for Sm resistance by colony plating and stepwise increase of Sm up to 25 micrograms/ml of culture medium. Several Sm-resistant colonies were obtained. Ribosomes were isolated from pEM109-transformed Sm-resistant and pKK3535-transformed Sm-sensitive cells. The ribosomes were assayed in vitro for Sm-induced misreading of poly(U) mRNA. We isolated 16S rRNA and sequenced the crucial RNA region by reverse transcription. The results clearly show that ribosomes from Sm-resistant cells correctly read the poly(U) mRNA in the presence of 25 micrograms Sm/ml of reaction mixture and the 16S rRNA contains the C912 to U transition. We conclude that C912 is involved in a translation step(s) which is (are) sensitive to streptomycin.