Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone.