PerlPrimer is a free, open-source GUI application written in Perl that designs primers for standard PCR, bisulphite PCR, real-time PCR (QPCR) and sequencing. It aims to automate and simplify the process of primer design. PerlPrimer's current features include the following: - Calculation of possible primer-dimers - Retrieval of genomic or cDNA sequences from Ensembl (including both sequences automatically for QPCR) - Ability to BLAST search primers using the NCBI server or a local server - Results can be saved or optionally exported in a tab-delimited format that is compatible with most spreadsheet applications. - ORF and CpG island detection algorithms - Ability to add cloning sequences to primers, automatically adjusted to be in-frame - QPCR primer design without manual intron-exon boundary entry An optional run-time dependency is spidey, for Real Time PCR. For details and citation: Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004 20(15):2471-2472