Abstract: The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1-Rho1-controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall-related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p-controlled cell wall genes and STRE-controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p-MAP kinase pathway, viz. Pkc1-R398A and Rho1-Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N)(10)GGC, was found. In Zymolyase -treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W)(4)TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway.CI - Copyright 2004 John Wiley & Sons, Ltd.