Next-generation sequencing technologies have revolutionized genome and transcriptome sequencing. RNA-Seq experiments are able to generate huge amounts of transcriptome sequence reads at a fraction of the cost of Sanger sequencing. Reads produced by these technologies are relatively short and error prone. To utilize such reads for transcriptome reconstruction and gene-structure identification, one needs to be able to accurately align the sequence reads over intron boundaries. In this unit, we describe PALMapper, a fast and easy-to-use tool that is designed to accurately compute both unspliced and spliced alignments for millions of RNA-Seq reads. It combines the efficient read mapper GenomeMapper with the spliced aligner QPALMA, which exploits read-quality information and predictions of splice sites to improve the alignment accuracy. The PALMapper package is available as a command-line tool running on Unix or Mac OS X systems or through a Web interface based on Galaxy tools.