BACKGROUND: Primer design is a critical step in all types of RT-PCR methods to ensure specificity and efficiency of a target amplicon. However, most traditional primer design programs suggest primers on a single template of limited genetic complexity. To provide researchers with a sufficient number of pre-designed specific RT-PCR primer pairs for whole genes in Arabidopsis, we aimed to construct a genome-wide primer-pair database. DESCRIPTION: We considered the homogeneous physical and chemical properties of each primer (homogeneity) of a gene, non-specific binding against all other known genes (specificity), and other possible amplicons from its corresponding genomic DNA or similar cDNAs (additional information). Then, we evaluated the reliability of our database with selected primer pairs from 15 genes using conventional and real time RT-PCR. CONCLUSION: Approximately 97% of 28,952 genes investigated were finally registered in AtRTPrimer. Unlike other freely available primer databases for Arabidopsis thaliana, AtRTPrimer provides a large number of reliable primer pairs for each gene so that researchers can perform various types of RT-PCR experiments for their specific needs. Furthermore, by experimentally evaluating our database, we made sure that our database provides good starting primer pairs for Arabidopsis researchers to perform various types of RT-PCR experiments.