Summary: Reconstructing transcript models from RNA-sequencing (RNA-seq) data and establishing these as independent transcriptional units can be a challenging task. The Zipper plot is an application that enables users to interrogate putative transcription start sites (TSSs) in relation to various features that are indicative for transcriptional activity. These features are obtained from publicly available datasets including CAGE-sequencing (CAGE-seq), ChIP-sequencing (ChIP-seq) for histone marks and DNase-sequencing (DNase-seq). The Zipper plot application requires three input fields (chromosome, genomic coordinate (hg19) of the TSS and strand) and generates a report that includes a detailed summary table, a Zipper plot and several statistics derived from this plot. Availability and Implementation: The Zipper plot is implemented using the statistical programming language R and is freely available at http://zipperplot.cmgg.be Contact: Pieter.Mestdagh@UGent.be; Katleen.DePreter@UGent.be; Francisco.AvilaCobos@UGent.be Supplementary information: Supplementary Methods available online.