SUMMARY: CrocoBLAST is a tool for dramatically speeding up BLAST+ execution on any computer. Alignments that would take days or weeks with NCBI BLAST+ can be run overnight with CrocoBLAST. Additionally, CrocoBLAST provides features critical for NGS data analysis, including: results identical to those of BLAST+; compatibility with any BLAST+ version; real-time information regarding calculation progress and remaining run time; access to partial alignment results; queueing, pausing, and resuming BLAST+ calculations without information loss. AVAILABILITY AND IMPLEMENTATION: CrocoBLAST is freely available online, with ample documentation (webchem.ncbr.muni.cz/Platform/App/CrocoBLAST). No installation or user registration is required. CrocoBLAST is implemented in C, while the graphical user interface is implemented in Java. CrocoBLAST is supported under Linux and Windows, and can be run under Mac OS X in a Linux virtual machine. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.