Many small, noncoding RNAs (sRNAs) in bacteria act as posttranscriptional regulators of messenger RNAs. TargetRNA2 is a web server that identifies mRNA targets of sRNA regulatory action in bacteria. As input, TargetRNA2 takes the sequence of an sRNA and the name of a sequenced bacterial replicon. When searching for targets of RNA regulation, TargetRNA2 uses a variety of features, including conservation of the sRNA in other bacteria, the secondary structure of the sRNA, the secondary structure of each candidate mRNA target and the hybridization energy between the sRNA and each candidate mRNA target. TargetRNA2 outputs a ranked list of likely regulatory targets for the input sRNA. When evaluated on a comprehensive set of sRNA-target interactions, TargetRNA2 was found to be both accurate and efficient in identifying targets of sRNA regulatory action. Furthermore, TargetRNA2 has the ability to integrate RNA-seq data, if available. If an sRNA is differentially expressed in two or more RNA-seq experiments, TargetRNA2 considers co-differential gene expression when searching for regulatory targets, significantly improving the accuracy of target identifications. The TargetRNA2 web server is freely available for use at http://cs.wellesley.edu/∼btjaden/TargetRNA2.