Binary complexes between messenger RNA and E. coli ribosomes were examined. A ribosome-mRNA binary complex on T4 gene 32 mRNA withstood inhibition by antibodies against ribosomal protein S1. Anti-S1 blocks ternary complex formation, as measured by "extension inhibition" or "toeprinting" analysis, only when preincubated with ribosomes prior to mRNA addition and not when anti-S1 was added after preincubation of ribosomes and mRNA. The ribosome was directly localized in a binary complex on two translation initiation sites by toeprinting analysis. In the absence of tRNA the ribosome halted cDNA synthesis by reverse transcriptase close to the Shine and Dalgarno sequence. Binary complex formation was inhibited by an oligodeoxynucleotide competitor of the Shine and Dalgarno sequence.