INTRODUCTION: The high complexity and dynamic nature of the regulation expression, protein synthesis, and protein activity pose a challenge to understand the cellular machinery. By deciphering the role of important including transcription factors, microRNAs, or small molecules, a better understanding of key regulatory processes can be obtained. Various contain information on the interactions of regulators with their targets different organisms, data recently being extended with the results of (Encyclopedia of DNA Elements) project. A systems biology approach our understanding on different regulators is essential in interpreting regulation of molecular biological processes. IMPLEMENTATION: We CyTargetLinker (http://projects.bigcat.unimaas.nl/cytargetlinker), a app, for integrating regulatory interactions in network analysis. released CyTargetLinker as one of the first apps for Cytoscape 3. It user-friendly and flexible interface to extend biological networks with regulatory interactions, such as microRNA-target, transcription factor- and/or drug-target. Importantly, CyTargetLinker employs identifier combine various interaction data resources that use different types of identifiers. RESULTS: Three case studies demonstrate the strength and applicability of CyTargetLinker, (i) extending a mouse molecular network, containing genes linked to diabetes mellitus, with validated predicted microRNAs, (ii) enriching a molecular interaction network, DNA repair genes, with ENCODE transcription factor and (iii) building a regulatory meta-network in which a biological process is extended with information on transcription factor, microRNA and drug regulation. CyTargetLinker provides a simple and extensible framework for biologists bioinformaticians to integrate different regulatory interactions into network analysis approaches. Visualization options enable biological interpretation of complex regulatory networks in a graphical way. incorporation of our tool into the Cytoscape framework allows the CyTargetLinker in combination with a wide variety of other apps for state-of-the-art network analysis.