The extracellular matrix (ECM) is a highly dynamic structure that not only provides a physical framework for cells within connective tissues, but also imparts instructive signals for development, tissue homeostasis and basic cell functions through its composition and ability to exert mechanical forces. The ECM of tissues is composed of, in addition to proteoglycans and hyaluronic acid, a number of proteins, most of which are generated after alternative splicing of their pre-mRNA. However, the precise function of these protein isoforms is still obscure in most cases. Fibronectin (FN), one of the main components of the ECM, is also one of the best-known examples of a family of proteins generated by alternative splicing, having at least 20 different isoforms in humans. Over the last few years, considerable progress on elucidating the functions of the alternatively spliced FN isoforms has been achieved with the essential development of key engineered mouse strains. Here we summarize the phenotypes of the mouse strains having targeted mutations in the FN gene, which may lead to novel insights linking function of alternatively spliced isoforms of fibronectin to human pathologies. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.