Gene expression studies employing high throughput real time PCR methods require finding uniform conditions for optimal amplification of multiple targets, often a daunting task. We developed a primer database, qPrimerDepot, which provides optimized primers for all human and mouse RefSeq genes. These primers are designed to amplify desired templates under unified annealing temperature. For most intron-bearing genes, primers flank one of the largest introns thus minimizing background noise due to genomic DNA contamination. The qPrimerDepot database can be accessed at http://primerdepot.nci.nih.gov/ and http://mouseprimerdepot.nci.nih.gov/.