The alpha-sarcin loop, located in domain VI of Escherichia coli 23S rRNA, is a universally conserved sequence involved in the binding of elongation factors to the ribosome and is the site of action of ribosome-inactivating proteins. Six mutations were created in this loop with the aim of establishing whether the mutant 23S rRNA could be assembled into functional ribosomes. In order to distinguish between plasmid-derived (mutant) and chromosome-derived (wild-type) 23S rRNAs, an oligonucleotide tag sequence was introduced into the plasmid-borne 23S rRNA gene. The tag sequence had no apparent effect on ribosome assembly or function. Two of the bases mutated (at positions A2660 and G2661) have been implicated in the binding of both elongation factor Tu and elongation factor G to the ribosome [Moazed, D., Robertson, J. M. & Noller, H. F. (1988) Nature 334, 362-364]. A further two bases (at positions C2658 and G2663) have been proposed to form a Watson-Crick base pair involved in the formation of a tetraloop structure required for ribosome function [Szewczak, A. A., Moore, P. B., Chan, Y. L. & Wool, I. G. (1993) Proc. Natl Acad. Sci. USA 90, 9581-9585]. It is inferred that the identity of the bases at positions 2658 and 2663 are of critical importance for ribosome structure and function, and that this function cannot be restored by a second mutation which potentially restores a Watson-Crick base pair, but with reversed position. Of five single mutants (each mutant containing one of the mutations C2658G, A2660G, G2661A, G2663C and G2664C) and one double mutant (containing both mutations C2658G and G2663C) only the two mutants with the single mutations G2661A and G2664C were incorporated into ribosomes at a level comparable to that of 23S rRNA expressed from a wild-type plasmid. However, the G2664C mutation resulted in a decrease in growth rate and a gradual loss of viability. rRNAs containing the G2663C single mutation and the C2658G and G2663C double mutation showed reduced incorporation into 50S subunits and these did not enter into ribosome couples.