The influence of 5'-secondary structure formation and 5'-leader length upon mRNA translation in yeast has been analyzed using a closely related set of cat mRNAs (Vega Laso, M. R., Zhu, D., Sagliocco, F. A., Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462). A cat mRNA with a relatively short unstructured 5'-leader (22 bases) had a ribosome loading about half that of a cat mRNA with an unstructured 5'-leader of 77 bases. The introduction of 5'-secondary structures at various positions throughout the 5'-leader of the cat mRNA inhibited translation initiation, the degree of inhibition being largely dependent upon the thermodynamic stability of the structure. Each mRNA carrying a 5'-secondary structure had a biphasic polysome distribution, indicating that the mRNA molecules were distributed between untranslated and well translated subpopulations. This suggests that once 5'-secondary structures are unwound, they reform slowly relative to the rate of translation initiation in yeast. Untranslated mRNA accumulated in 43 S preinitiation complexes, even when there were only 5 bases between the 5'-cap and the base of the hairpin. The data are consistent with the scanning hypothesis (Kozak, M. (1989) J. Cell. Biol. 108, 229-241) and suggest that 40 S ribosomal subunits bind to mRNA early in the scanning process, probably before mRNA unwinding has taken place.