PDB2PQR is a web server that automates the preparation of biomolecular structures for molecular simulations. It addresses the need for accurate electrostatic field calculations by converting PDB structures into formats compatible with various computational biology tools. The updated version of PDB2PQR includes robust command line support, improved pKa estimation via PROPKA, ligand parameterization via PEOE_PB charge methodology, an expanded set of force fields, and user-defined parameters via XML input files. These features are available through a new web interface (http://pdb2pqr.sourceforge.net/). The server facilitates the setup and execution of continuum electrostatics calculations, including adding missing heavy atoms, estimating titration states, assigning charge and radius parameters, and generating PQR output. It supports a wide range of computational biology applications, including docking, simulation, and visualization. The PDB2PQR web service is driven by a modular, Python-based collection of routines, providing flexibility for non-interactive, high-throughput usage. It is available via multiple web mirrors and allows users to customize output with various atom naming schemes. The server also includes a 'map' that presents a table of atoms' name/number, residue name, chain name, AMBER atom type, and CHARMM atom type to aid in parameter assignment. Ligand parameterization requires the user to specify the ligand structure, protonation state, and formal charge in the MOL2 format. The calculation of ligand charges is based on the PEOE procedure, which has been optimized for better agreement between theoretical and experimental solvation energies. The current version of PDB2PQR supports an 'extension' directory for user-defined processing of output. Future developments include a pdb2mol2 parser, correct treatment of protein posttranslational modification, and integration of a Poisson–Boltzmann continuum electrostatics-based pKa calculation algorithm. The PDB2PQR service is expected to remain a valuable tool for the structural and computational biology communities.PDB2PQR is a web server that automates the preparation of biomolecular structures for molecular simulations. It addresses the need for accurate electrostatic field calculations by converting PDB structures into formats compatible with various computational biology tools. The updated version of PDB2PQR includes robust command line support, improved pKa estimation via PROPKA, ligand parameterization via PEOE_PB charge methodology, an expanded set of force fields, and user-defined parameters via XML input files. These features are available through a new web interface (http://pdb2pqr.sourceforge.net/). The server facilitates the setup and execution of continuum electrostatics calculations, including adding missing heavy atoms, estimating titration states, assigning charge and radius parameters, and generating PQR output. It supports a wide range of computational biology applications, including docking, simulation, and visualization. The PDB2PQR web service is driven by a modular, Python-based collection of routines, providing flexibility for non-interactive, high-throughput usage. It is available via multiple web mirrors and allows users to customize output with various atom naming schemes. The server also includes a 'map' that presents a table of atoms' name/number, residue name, chain name, AMBER atom type, and CHARMM atom type to aid in parameter assignment. Ligand parameterization requires the user to specify the ligand structure, protonation state, and formal charge in the MOL2 format. The calculation of ligand charges is based on the PEOE procedure, which has been optimized for better agreement between theoretical and experimental solvation energies. The current version of PDB2PQR supports an 'extension' directory for user-defined processing of output. Future developments include a pdb2mol2 parser, correct treatment of protein posttranslational modification, and integration of a Poisson–Boltzmann continuum electrostatics-based pKa calculation algorithm. The PDB2PQR service is expected to remain a valuable tool for the structural and computational biology communities.