MOTIVATION: Identification of differential expressed genes has led to countless new discoveries. However, differentially expressed genes are only a proxy for finding dysregulated pathways. The problem is to identify how the network of regulatory and physical interactions rewires in different conditions or in disease. RESULTS: We developed a procedure named DINA (DIfferential Network Analysis), which is able to identify set of genes, whose co-regulation is condition-specific, starting from a collection of condition-specific gene expression profiles. DINA is also able to predict which transcription factors (TFs) may be responsible for the pathway condition-specific co-regulation. We derived 30 tissue-specific gene networks in human and identified several metabolic pathways as the most differentially regulated across the tissues. We correctly identified TFs such as Nuclear Receptors as their main regulators and demonstrated that a gene with unknown function (YEATS2) acts as a negative regulator of hepatocyte metabolism. Finally, we showed that DINA can be used to make hypotheses on dysregulated pathways during disease progression. By analyzing gene expression profiles across primary and transformed hepatocytes, DINA identified hepatocarcinoma-specific metabolic and transcriptional pathway dysregulation. AVAILABILITY: We implemented an on-line web-tool http://dina.tigem.it enabling the user to apply DINA to identify tissue-specific pathways or gene signatures. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.