The decoding region of 16 S rRNA (nucleotides 1396-1403) is not available to complementary DNA oligomer binding in 70 S ribosomes, despite high levels of binding of these oligomers to active 30 S ribosomal subunits. While addition of poly(U) or tRNA(Phe) alone did not affect the binding of either cDNA to 30 or 70 S ribosomes, poly(U)-directed tRNA(Phe) decreased the binding of cDNA (1396-1403) oligomers by 80% to the active 30 S ribosomal subunits. But the poly(U)-directed tRNA(Phe) cannot displace a prebound cDNA in this region, nor can the cDNA displace a prebound poly(U)-directed tRNA(Phe). A cDNA to the anti-Shine-Dalgarno region (nucleotides 1534-1541) binds equally well to 30 and 70 S ribosomal particles, but the simultaneous addition of poly(U)/tRNA(Phe) and cDNA results in a 50% decrease in binding of the cDNA. In this case the poly(U)-directed tRNA(Phe) does displace the prebound cDNA, but the cDNA cannot displace a prebound poly(U)-directed tRNA(Phe). Neither cDNA oligomer inhibits the reassociation of the ribosomal subunits into 70 S particles.