The article by Triinu Koressaar and Mado Remm discusses enhancements and modifications to the primer design program Primer3. The key improvements include a new formula for calculating melting temperature (Tm) and a salt correction formula that accounts for divalent cations, which are common in PCR buffers. The modified version also allows for the use of lowercase masked template sequences, enabling primer design that can overlap masked regions. The new features have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer). A modified version called mPrimer3 is also available independently, with a web-based version accessible at http://bioinfo.ebc.ee/primer3/ and the binary code freely downloadable from http://bioinfo.ebc.ee/download/. The article provides detailed mathematical formulations for the new Tm calculation methods and compares their performance with the original Primer3 version, showing significant improvements in accuracy.The article by Triinu Koressaar and Mado Remm discusses enhancements and modifications to the primer design program Primer3. The key improvements include a new formula for calculating melting temperature (Tm) and a salt correction formula that accounts for divalent cations, which are common in PCR buffers. The modified version also allows for the use of lowercase masked template sequences, enabling primer design that can overlap masked regions. The new features have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer). A modified version called mPrimer3 is also available independently, with a web-based version accessible at http://bioinfo.ebc.ee/primer3/ and the binary code freely downloadable from http://bioinfo.ebc.ee/download/. The article provides detailed mathematical formulations for the new Tm calculation methods and compares their performance with the original Primer3 version, showing significant improvements in accuracy.