<jats:title>Summary</jats:title><jats:p>To investigate the changes in profiles of mRNA accumulation in response to sulfur deficiency, approximately 13 000 non‐redundant <jats:italic>Arabidopsis thaliana</jats:italic> ESTs corresponding to approximately 9000 genes were analyzed using DNA macroarray. Three‐week‐old <jats:italic>Arabidopsis</jats:italic> plants grown on an agarose‐solidified control medium were transferred to a sulfate‐free medium and grown for 48 h for the analyses of sulfur‐related metabolites and global gene expression profiles. Concentrations of sulfate, <jats:italic>O</jats:italic>‐acetyl‐<jats:sc>l</jats:sc>‐serine (OAS), a positive regulator of sulfur deficiency‐responsive genes, cysteine and glutathione (GSH) were determined. Plants transferred to sulfate‐free media had reduced concentrations of sulfate and GSH, and OAS concentrations increased. Macroarray analysis revealed a number of genes, including <jats:italic>APR2</jats:italic> and <jats:italic>Sultr1;2</jats:italic>, whose mRNA accumulation was increased by sulfur deficiency. Profiling was also carried out with plants treated with OAS under sulfate‐sufficient condition. Scatter plot analysis revealed a positive correlation between the changes of expression levels by sulfur deficiency and by OAS treatment among the clones tested, suggesting that mRNA accumulation of a number of genes under sulfur deficiency is mainly controlled by OAS concentrations in tissues. It was also revealed that the sets of genes regulated under sulfur deficiency in leaves and roots differ considerably.