A vector containing the leftward promoter (pL) as transcription initiation signal and a synthetic, easily adaptable translation initiation region have been constructed. We have used the expression system to assess the relevance of sequences upstream from the Shine-Dalgarno (SD) region in the translational-initiation process. To this end, a series of structural variants of the prototype ribosome-binding site were used to direct the synthesis of both mature human fibroblast interferon and beta-galactosidase (beta-gal). It was found that alterations 5' to the SD element can considerably affect the rate of mRNA translation. The observation that the relative efficiency of the various 5'-untranslated regions depends on the downstream coding information implies that secondary (and/or tertiary) structure formation is of major importance in the initiation process. But an mRNA folding, in which the SD and ATG determinant are set free in single-stranded regions, does not unconditionally guarantee an efficient initiation of translation.