Abstract Motivation: Many RNA molecules function without being translated into proteins, and function depends on structure. Pseudoknots are motifs in RNA secondary structures that are difficult to predict but are also often functionally important. Results: TurboKnot is a new algorithm for predicting the secondary structure, including pseudoknotted pairs, conserved across multiple sequences. TurboKnot finds 81.6% of all known base pairs in the systems tested, and 75.6% of predicted pairs were found in the known structures. Pseudoknots are found with half or better of the false-positive rate of previous methods. Availability: The program is available for download under an open-source license as part of the RNAstructure package at: http://rna.urmc.rochester.edu. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.