Motivations. Next-generation sequencing (NGS) is producing large data volumes at reasonable cost and new applications are being developed at increasing speed. A common denominator for all applications of NGS technology is the need to annotate genomic regions of interest. Tools such as Galaxy [1], CisGenome [2], or the Bioconductor ChIPpeakAnno package [3] have been published to perform this task. However, using these tools often requires a significant amount of bioinformatics skills and/or downloading and installing dedicated software. A widely accepted, webbased annotation tool available to bioinformaticians and biologists with widely varying skill levels is not available. Indeed, many skilled bioinformaticians rely on self-made scripts to process the data to be annotated in the desired input/output format and in the necessary detail. For many biologists working with new generation sequencing data, annotating a set of genomic regions represents a complicated task that necessarily involves the help of a skilled bioinformatician. Methods. Here we present AnnotateGenomicRegions, a web application that accepts genomic regions as input and outputs overlapping and/or neighboring genome annotations chosen on a simple web-form. The application is based on Java Enterprise technology and runs on a Glassfish server. The necessary speed of annotating hundreds of thousands of genomic regions with tens of different annotations within seconds is achieved using a proprietary hash-based data structure. Results. We developed an annotation tool that fulfills five basic design criteria: 1. genomic regions shall be used as input query; 2. the output shall be pastable into an Excel table; 3. the application shall be web-based; 4. no programming skills required to use the application; 5. it must be fast enough to annotate hundreds of thousands of genomic regions within seconds. The tool can be installed on any computer capable of running Java and Glassfish on a Windows or Unix/Linux operating system, which is from a laptop to a mainframe computer. Availability http://sourceforge.net/project/annotatelocus/?source=directory References 1. Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, et al. (2005) Galaxy: a platform for interactive largescale genome analysis. Genome Res 15:1451-5 2. Ji H, Jiang H, Ma W, Johnson DS, Myers RM, Wong WH (2008) An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat Biotechnol. 26:1293-300 3. Zhu LJ, Gazin C, Lawson ND, Pages H, Lin SM, Lapointe DS, et al. (2010) ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 11:237