Recombinant DNA and classic genetic procedures were used to map a spectinomycin resistance mutation to a 121 base pair region of a 16S RNA gene and a macrolide-lincosamide-streptogramin type B resistance mutation to a 32 base pair region of a 23S RNA gene. DNA sequence analysis of these regions revealed that spectinomycin resistance results from a C/G to T/A transition at position 1192 of a 16S RNA gene. Resistance to macrolide, lincosamide and streptogramin type B antibiotics results from an A/T to T/A transversion at position 2058 of a 23S RNA gene. The alteration in 16S RNA is in a sequence that can participate in alternate base pairing arrangements that have been proposed to be involved in the translocation process. The alteration in 23S RNA identifies sequences important to peptidyl transfer.