Primer3 is a widely used program for designing PCR primers. This paper describes several improvements to Primer3, including a new formula for calculating melting temperature (Tm) and a salt correction formula. The new version also accounts for the effects of divalent cations, which are commonly present in PCR buffers. Another modification allows the use of lowercase-masked template sequences for primer design. These features have been implemented into the development code of Primer3 and are available in future versions (version 1.1 and newer). A modified version, called mPrimer3, is also available independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/. The original version of Primer3 used a table of thermodynamic parameters and a formula for Tm calculations that are now considered outdated. Improved sets of thermodynamic parameters and formulas are now available. The melting temperature of a primer is influenced by the concentration of mono- and divalent cations in the solution. The original Primer3 uses a salt correction formula that does not consider the effect of divalent cations. Many other primer design programs also use outdated formulas for Tm calculation. However, some programs already use up-to-date thermodynamic parameters for melting temperature calculation. However, few of these programs allow automatic command-line calculations for large datasets. Another problem with the current version of Primer3 is that it does not support the use of soft-masked (lowercase-masked) target sequences. To address this, a new feature has been implemented in mPrimer3 that allows primers to be designed from lowercase-masked sequences. Lowercase masking preserves the DNA sequence and allows primers to be designed that partly overlap the masked region. The paper also describes two different salt correction formulas for melting temperature calculations. The first is a sequence-independent equation, and the second is a sequence-dependent equation. The results of comparing experimentally measured melting temperatures with Tm values predicted by the original Primer3 and two different formulae in modified Primer3 are shown in Figure 1. The paper also describes a new feature in Primer3 that allows primers overlapping lowercase-masked regions to be designed. A novel feature of the modified Primer3 is that primers with a lowercase nucleotide at the 3' end can be rejected. This behavior relies on the assumption that masked features can partly overlap the primer, but they cannot overlap its 3' end. Lowercase letters in other positions are accepted, assuming that the masked features do not influence primer performance if they do not overlap the 3' end.Primer3 is a widely used program for designing PCR primers. This paper describes several improvements to Primer3, including a new formula for calculating melting temperature (Tm) and a salt correction formula. The new version also accounts for the effects of divalent cations, which are commonly present in PCR buffers. Another modification allows the use of lowercase-masked template sequences for primer design. These features have been implemented into the development code of Primer3 and are available in future versions (version 1.1 and newer). A modified version, called mPrimer3, is also available independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/. The original version of Primer3 used a table of thermodynamic parameters and a formula for Tm calculations that are now considered outdated. Improved sets of thermodynamic parameters and formulas are now available. The melting temperature of a primer is influenced by the concentration of mono- and divalent cations in the solution. The original Primer3 uses a salt correction formula that does not consider the effect of divalent cations. Many other primer design programs also use outdated formulas for Tm calculation. However, some programs already use up-to-date thermodynamic parameters for melting temperature calculation. However, few of these programs allow automatic command-line calculations for large datasets. Another problem with the current version of Primer3 is that it does not support the use of soft-masked (lowercase-masked) target sequences. To address this, a new feature has been implemented in mPrimer3 that allows primers to be designed from lowercase-masked sequences. Lowercase masking preserves the DNA sequence and allows primers to be designed that partly overlap the masked region. The paper also describes two different salt correction formulas for melting temperature calculations. The first is a sequence-independent equation, and the second is a sequence-dependent equation. The results of comparing experimentally measured melting temperatures with Tm values predicted by the original Primer3 and two different formulae in modified Primer3 are shown in Figure 1. The paper also describes a new feature in Primer3 that allows primers overlapping lowercase-masked regions to be designed. A novel feature of the modified Primer3 is that primers with a lowercase nucleotide at the 3' end can be rejected. This behavior relies on the assumption that masked features can partly overlap the primer, but they cannot overlap its 3' end. Lowercase letters in other positions are accepted, assuming that the masked features do not influence primer performance if they do not overlap the 3' end.