We describe the development of a high-density Arabidopsis‘whole genome’ oligonucleotide probe array for expression analysis (the Affymetrix ATH1 GeneChip® probe array) that contains approximately 22 750 probe sets. Precedence on the array was given to genes for which either expression evidence or a credible database match existed. The remaining space was filled with ‘hypothetical’ genes. The new ATH1 array represents approximately 23 750 genes of which 60% were detected in RNA from cultured seedlings. Sensitivity of the array, determined using spiking controls, was approximately one transcript per cell. The array demonstrated high technical reproducibility and concordance with real-time PCR results. Indole-3 acetic acid (IAA)-induced changes in gene expression were used for biological validation of the array. A total of 222 genes were significantly upregulated and 103 significantly downregulated by exposure to IAA. Of the genes whose products could be functionally classified, the largest specific classes of upregulated genes were transcriptional regulators and protein kinases, many fewer of which were represented among the downregulated genes. Over one-third of the auxin-regulated genes have no known function, although many belong to gene families with members that have previously been shown to be auxin regulated. For the 6714 genes represented both on this and the earlier Arabidopsis Genome (AG) array, both signal intensities and gene expression ratios were very similar. Mapping of the oligonucleotides on the ATH1 array to the latest (version 4.0) annotation showed that over 95% of the probe sets (based on version 2.0 annotation) still fully represented their original target genes.