PINCH2 belongs, together with PINCH1, to a new family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain, with the integrin-linked kinase and thereby link integrins with several signal transduction pathways. Despite their high similarity, it has been shown that PINCH1 and PINCH2 could exert distinct functions during cell spreading and cell survival. To investigate the function of PINCH2 in vivo, we deleted PINCH2 in mouse using the loxP/Cre system. In contrast to the PINCH1-deficient mice, which die at the peri-implantation stage, PINCH2-null mice are viable, fertile and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder and kidney revealed no apparent abnormalities, but showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, overlapping function in vivo. To further test this possibility, we established PINCH1-null mouse embryonic fibroblasts, which express neither PINCH1 nor PINCH2. We found that in fibroblasts with a PINCH1/2-null background, PINCH2 is able to rescue the spreading and adhesion defects of mutant fibroblasts to the same extent as PINCH1. Furthermore, we show that the LIM1 domain only of either PINCH1 or PINCH2 can prevent ILK degradation despite their failure to localize to focal adhesions. Altogether these results suggest that PINCH1 and PINCH2 share overlapping functions and operate dependently and independently of their subcellular localization.