PubChem (http://pubchem.ncbi.nlm.nih.gov) is a public repository for biological properties of small molecules hosted by the US National Institutes of Health (NIH). PubChem BioAssay database currently contains biological test results for more than 700 000 compounds. The goal of PubChem is to make this information easily accessible to biomedical researchers. In this work, we present a set of web servers to facilitate and optimize the utility of biological activity information within PubChem. These web-based services provide tools for rapid data retrieval, integration and comparison of biological screening results, exploratory structure-activity analysis, and target selectivity examination. This article reviews these bioactivity analysis tools and discusses their uses. Most of the tools described in this work can be directly accessed at http://pubchem.ncbi.nlm.nih.gov/assay/. URLs for accessing other tools described in this work are specified individually.