The downstream box (db) with complementarity to a segment in the penultimate stem of 16S rRNA has been suggested to serve as a recognition element for the ribosome. For some mRNAs, the db has been proposed to act synergistically with the Shine and Dalgarno sequence (SD), while for the leaderless lambda(cI) mRNA it has been suggested to substitute for the SD in translation initiation. To test whether the db-anti-db interaction is required for translation initiation, we have used three different leaderless transcripts, the lambda(cI), phage P2 gene V and Tn1721 tetR mRNA. Using primer extension inhibition analysis (toeprinting), we show that the db does not influence translation initiation complex formation in vitro. In an attempt to demonstrate the simultaneous interactions between the db and anti-db and between the anticodon of initiator tRNA and the start codon, respectively, chemical probing has been employed on cI translation initiation complexes. These studies did not reveal a protection of the bases comprising the putative db in cI. In addition, kinetic toeprinting experiments and in vivo expression studies with cI mRNA showed that the db is dispensable for the initial interaction between ribosome and cI mRNA in the pathway towards formation of the initiation complex.