PyNAST is a flexible and portable reimplementation of the Nearest Alignment Space Termination (NAST) tool, originally developed for aligning 16S rRNA genes in microbial community analysis. The new tool includes three user-friendly interfaces: a Mac OS X GUI, a command-line interface, and an application programming interface (API). PyNAST allows users to align sequences to arbitrary template alignments, enhancing its applicability beyond microbial community analysis. Key features include parameterized algorithms, open-source licensing, and minimal dependencies. The algorithm ensures that aligned sequences are the same length as the template, facilitating the integration of new sequences into existing alignments. Performance benchmarks show that PyNAST runs faster than the original NAST implementation, with a per-sequence runtime of 1.46 seconds compared to 1.55 seconds. PyNAST's enhanced portability and flexibility make it a valuable tool for analyzing larger datasets and novel applications.PyNAST is a flexible and portable reimplementation of the Nearest Alignment Space Termination (NAST) tool, originally developed for aligning 16S rRNA genes in microbial community analysis. The new tool includes three user-friendly interfaces: a Mac OS X GUI, a command-line interface, and an application programming interface (API). PyNAST allows users to align sequences to arbitrary template alignments, enhancing its applicability beyond microbial community analysis. Key features include parameterized algorithms, open-source licensing, and minimal dependencies. The algorithm ensures that aligned sequences are the same length as the template, facilitating the integration of new sequences into existing alignments. Performance benchmarks show that PyNAST runs faster than the original NAST implementation, with a per-sequence runtime of 1.46 seconds compared to 1.55 seconds. PyNAST's enhanced portability and flexibility make it a valuable tool for analyzing larger datasets and novel applications.