Alternative Splicing (AS) is the molecular phenomenon whereby multiple transcripts are produced from the same gene locus. As a consequence, it is responsible for the expansion of eukaryotic transcriptomes. Aberrant AS is involved in the onset and progression of several human diseases. Therefore, the characterization of exon-intron structure of a gene and the detection of corresponding transcript isoforms is an extremely relevant biological task. Nonetheless, the computational prediction of AS events and the repertoire of alternative transcripts is yet a challenging issue. Hereafter we introduce PIntron, a software package to predict the exon-intron structure and the full-length isoforms of a gene given a genomic region and a set of transcripts (ESTs and/or mRNAs). The software is open source and available at http://pintron.algolab.eu. PIntron has been designed for (and extensively tested on) a standard workstation without requiring dedicated expensive hardware. It easily manages large genomic regions and more than 20,000 ESTs, achieving good accuracy as shown in an experimental evaluation performed on 112 well-annotated genes selected from the ENCODE human regions used as training set in the EGASP competition.