PerlPrimer is a cross-platform application for the design of standard PCR, bisulfite PCR, real-time PCR, and sequencing primers. The program combines accurate primer-design algorithms with powerful interfaces to commonly used Internet databases, such as sequence retrieval from the Ensembl genome databases and the ability to BLAST search primer pairs. The use of PerlPrimer for designing primers is described, together with a synopsis of the primer search and primer-dimer algorithms.