The differentiation of 3T3-F442A cells is accompanied by changes in cell morphology, decreased synthesis and assembly of cytoskeletal proteins, and increased biosynthesis of lipogenic enzymes. The differentiation-dependent alterations in actin assembly can be prevented by culture of cells on fibronectin matrices. Under these conditions, morphological change is prevented and the induction of lipogenic enzymes such as glycerophosphate dehydrogenase and fatty acid synthetase is sharply reduced. In contrast, the differentiation-linked decreases in actin and tubulin biosynthesis occur normally. Hybridization of cytoplasmic and nuclear RNA to cloned cDNA probes indicates that the inhibition of lipogenic protein biosynthesis occurs because of reduced levels of specific mRNA production. The inhibitory effects of fibronectin can be reversed by keeping cells in a rounded configuration or by exposing cells to either cytochalasin D, which disrupts the actin cytoskeleton, or insulin, a key lipogenic hormone. These results demonstrate that fibronectin can regulate gene expression for lipogenic proteins and they suggest that it interferes with cytoskeletal and morphological changes necessary for new gene expression.