Primer3 is a widely used open-source software for designing PCR primers, with significant updates and enhancements over the past decade. The latest version improves primer design by incorporating more accurate thermodynamic models for predicting melting temperatures, reducing the likelihood of primer hairpins or dimers, and enhancing specificity through whole-genome sequence analysis. It also allows for precise control of primer placement and includes features to save and reuse parameter settings, ensuring primers are not used in multiple primer pairs. The core code has been made more modular, and programming interfaces have been improved for easier integration with other software. These improvements position Primer3 for continued use in genome-scale data analysis. Primer3 is available as a command-line program, a web interface (Primer3web), and a web service (Primer3Plus). It supports various design tasks, including PCR primer design, checking existing primers, and generating lists of legal primers. The software also includes a programming library (libprimer3) for direct integration with other programs. New features include enhanced thermodynamic models for predicting primer behavior, improved primer location control, and a new search algorithm for more efficient primer selection. Primer3 has been integrated into numerous software packages and web services, including PRIMEGENS, the PCR suite, and Primer-BLAST. These tools leverage Primer3's capabilities to enhance primer design for specific applications such as methylation analysis, splice variant profiling, and SNP typing. The software also supports the design of primers for RT-PCR and ensures that primers span exon-exon junctions to avoid amplifying genomic DNA or unspliced transcripts. The usability of Primer3 has been improved with features such as the ability to store and reuse argument settings, rationalized boulder IO tags, and a more user-friendly web interface. These enhancements make Primer3 more accessible to both expert and occasional users. The software is now more efficient and flexible, with improved integration capabilities and a broader range of applications in bioinformatics, genomics, and molecular biology.Primer3 is a widely used open-source software for designing PCR primers, with significant updates and enhancements over the past decade. The latest version improves primer design by incorporating more accurate thermodynamic models for predicting melting temperatures, reducing the likelihood of primer hairpins or dimers, and enhancing specificity through whole-genome sequence analysis. It also allows for precise control of primer placement and includes features to save and reuse parameter settings, ensuring primers are not used in multiple primer pairs. The core code has been made more modular, and programming interfaces have been improved for easier integration with other software. These improvements position Primer3 for continued use in genome-scale data analysis. Primer3 is available as a command-line program, a web interface (Primer3web), and a web service (Primer3Plus). It supports various design tasks, including PCR primer design, checking existing primers, and generating lists of legal primers. The software also includes a programming library (libprimer3) for direct integration with other programs. New features include enhanced thermodynamic models for predicting primer behavior, improved primer location control, and a new search algorithm for more efficient primer selection. Primer3 has been integrated into numerous software packages and web services, including PRIMEGENS, the PCR suite, and Primer-BLAST. These tools leverage Primer3's capabilities to enhance primer design for specific applications such as methylation analysis, splice variant profiling, and SNP typing. The software also supports the design of primers for RT-PCR and ensures that primers span exon-exon junctions to avoid amplifying genomic DNA or unspliced transcripts. The usability of Primer3 has been improved with features such as the ability to store and reuse argument settings, rationalized boulder IO tags, and a more user-friendly web interface. These enhancements make Primer3 more accessible to both expert and occasional users. The software is now more efficient and flexible, with improved integration capabilities and a broader range of applications in bioinformatics, genomics, and molecular biology.