AbstractMotivation: Since 1990, the basic local alignment search tool (BLAST) has become one of the most popular and fundamental bioinformatics tools for sequence similarity searching, receiving extensive attention from the research community. The two pioneering papers on BLAST have received over 96 000 citations. Given the huge population of BLAST users and the increasing size of sequence databases, an urgent topic of study is how to improve the speed. Recently, graphics processing units (GPUs) have been widely used as low-cost, high-performance computing platforms. The existing GPU-BLAST is a promising software tool that uses a GPU to accelerate protein sequence alignment. Unfortunately, there is still no GPU-accelerated software tool for BLAST-based nucleotide sequence alignment.Results: We developed G-BLASTN, a GPU-accelerated nucleotide alignment tool based on the widely used NCBI-BLAST. G-BLASTN can produce exactly the same results as NCBI-BLAST, and it has very similar user commands. Compared with the sequential NCBI-BLAST, G-BLASTN can achieve an overall speedup of 14.80X under ‘megablast’ mode. More impressively, it achieves an overall speedup of 7.15X over the multithreaded NCBI-BLAST running on 4 CPU cores. When running under ‘blastn’ mode, the overall speedups are 4.32X (against 1-core) and 1.56X (against 4-core). G-BLASTN also supports a pipeline mode that further improves the overall performance by up to 44% when handling a batch of queries as a whole. Currently G-BLASTN is best optimized for databases with long sequences. We plan to optimize its performance on short database sequences in our future work.Availability: http://www.comp.hkbu.edu.hk/∼chxw/software/G-BLASTN.htmlContact: chxw@comp.hkbu.edu.hkSupplementary information: Supplementary data are available at Bioinformatics online.