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Background  High-throughput screening of protein-protein interactions opens new systems biology perspectives for the comprehensive understanding of cell physiology in normal and pathological conditions. In this context, yeast two-hybrid system appears as a promising approach to efficiently reconstruct protein interaction networks at the proteome-wide scale. This protein interaction screening method generates a large amount of raw sequence data,  i.e . the ISTs (Interaction Sequence Tags), which urgently need appropriate tools for their systematic and standardised analysis.    Findings  We develop pISTil, a bioinformatics pipeline combined with a user-friendly web-interface: ( i ) to establish a standardised system to analyse and to annotate ISTs generated by two-hybrid technologies with high performance and flexibility and ( ii ) to provide high-quality protein-protein interaction datasets for systems-level approach. This pipeline has been validated on a large dataset comprising more than 11.000 ISTs. As a case study, a detailed analysis of ISTs obtained from yeast two-hybrid screens of Hepatitis C Virus proteins against human cDNA libraries is also provided.    Conclusion  We have developed pISTil, an open source pipeline made of a collection of several applications governed by a Perl script. The pISTil pipeline is intended to laboratories, with IT-expertise in system administration, scripting and database management, willing to automatically process large amount of ISTs data for accurate reconstruction of protein interaction networks in a systems biology perspective. pISTil is publicly available for download at   .

pISTil: a pipeline for yeast two-hybrid Interaction Sequence Tags identification and analysis