AbstractThe budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway. Using lacZ–reporter gene fusions, clonal cell lines were screened for levels of β‐galactosidase activity in the presence or absence of osmotic stress. A screen of 9000 transformants displayed 663 (7%) gene fusions that were active in rich medium. Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening. The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis. Five stress‐activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1. The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane. Copyright © 2003 John Wiley & Sons, Ltd.