MOTIVATION: Improving the usability of bioinformatics resources enables researchers to find, interact with, share, compare and manipu-late important information more effectively and efficiently. It thus enables researchers to gain improved insights into biological processes with the potential, ultimately, of yielding new scientific results. Usability 'barriers' can pose significant obstacles to a satisfactory user experience and force researchers to spend unnecessary time and effort to complete their tasks. The number of online biological databases available is growing and there is an expanding community of diverse users. In this context there is an increasing need to ensure the highest stan-dards of usability. RESULTS: Using 'state-of-the-art' usability evaluation methods, we have identified and characterised a sample of usability issues potentially relevant to web bioinformatics resources in general. These specifically concern the design of the navigation and search mechanisms available to the user. The usability issues we have discovered in our substantial case studies are un-dermining the ability of users to find the information they need in their daily research activities. In addition to characterising these issues, specific recommendations for improvements are proposed leveraging proven practices from web and usability engineering. The methods and approach we exemplify can be readily adopted by the developers of bioinformatics resources. Supplementary Information: additional data about the usability methods and a summary of the results of the usability analyses presented in the paper is provided in a supplementary infor-mation file.