The p130 protein shares extensive sequence similarity with pRB, the product of the retinoblastoma gene, and is a major E2F-associated protein in quiescent cells. To investigate its biological function, we have mutated p130 via gene targeting in the mouse. Homozygous mutation of p130 had little discernible effect on development or on the growth of mouse embryo fibroblasts in culture. Much of the E2F activity that normally associates with p130 in serum-starved mouse embryo fibroblasts associated instead with the highly related p107 protein. To determine whether p130 and p107 have overlapping biological roles, we produced mice having simultaneous inactivation of the p130 and p107 genes. Such mice exhibited deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal lethality. These findings indicate that p130 and p107 play an important role in limb development through their abilities to control chondrocyte proliferation. Thus, in certain settings p107 and p130 perform growth-regulatory functions that are not fulfilled by pRB.