A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae. We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady-state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9. mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure. A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98%. A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66%. The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader. An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined. Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo. The stem-loop structures were far less inhibitory in a reticulocyte lysate system. Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression.