Primer Design Assistant (PDA) is a web interface primer design service combined with thermodynamic theory to evaluate the fitness of primers. It runs in a Linux-Apache-MySQL-PHP structure on a PC equipped with dual CPU (Intel Pentium III 1.4 GHz) and 512 Mb of RAM. A succinct user interface of PDA is accomplished by built-in parameters setting. Advanced options on 5' GC content, 3' GC content, dimer check and hairpin check are available. The option of covered region constrains the PCR product to cover a user-defined segment. PDA accepts single sequence query or multiple ones in FASTA format. It produces optimal and homogenous primer pairs that meet the need in experimental design with large-scaled PCR amplifications. Considering the system loading, the size of a submitted sequence is limited to 10 kb and the total sequence number in a query is limited to 20. The authors may be contacted regarding other requirements for primer design. The web application can be found at http://dbb.nhri.org.tw/primer/.