Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitable in vitro translation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-free Saccharomyces cerevisiae lysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5' cap and a 3' poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events. (C) 1997 Academic Press.