The actins are essential cytoskeletal proteins required for the survival and growth of cells. The transitions between soluble (G-actin) and filamentous (F-actin) forms of actin (actin treadmilling) are complexly regulated. Here we show that the expression of the cytoplasmic beta-actin and gamma-actin genes is down-regulated in mouse fibroblasts when the cell density of the culture increases. Conversely, a dense culture replated at lower density results in increases in actin mRNA levels within a few hours. Concomitant with these changes in mRNA levels, we observe increased depolymerization of actin microfilaments at higher densities resulting in an elevated G-actin to F-actin ratio. By using actin polymerization inhibitors, we show that the density-dependent change in actin gene expression is dependent on changes in the ratio of G-actin vs. F-actin levels. Therefore, actin treadmilling and actin gene regulation are not coregulated by cell density, but represent a linear signal transduction pathway in which actin treadmilling regulates actin gene transcription. The physiological transition represented by the growth of a sparse fibroblast population into a confluent and growth-arrested population represents a useful model for the study of how the actin treadmill exerts its action on the gene expression program of cells.