We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G 1 phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of ␣51 integrin. Mechanistically, ␣51 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G 1 phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominantnegative Rho kinase led to a more rapid progression through G 1 phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21 cip1 and p27 kip1 , were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G 1 phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G 1 phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.Much like cells deprived of growth factors or attachment to an extracellular matrix (ECM), fibroblasts cultured under conditions that preclude cell spreading become arrested in G 1 phase of the cell cycle (14,21,26,33,36). The cyclin-dependent kinases (cdk's), cyclin D-cdk4 (or cdk6) and cyclin Ecdk2, are the key regulators of cell cycle progression through G 1 phase, and several studies now indicate that disruption of cell spreading prevents the activation of these enzymes. Pharmacological inhibitors of actin polymerization block the induction of cyclin D1 (typically the rate-limiting step in formation of active cyclin D1-cdk4 or -cdk6 complexes), the downregulation of p21 cip1 and p27 kip1 (inhibitors of cyclin E-cdk2), phosphorylation of the retinoblastoma protein, and entry into S phase (10,11,22,31,32). In endothelial cells, spreading is associated with the translation of cyclin D1 mRNA, the downregulation of p27 kip1 , and S phase entry (31, 44). Similarly, when fibroblasts are cultured within collagen gels, the disruption of isometric tension leads to loss of actin stress fibers, the inactivation of extracellular signal-regulated kinases (ERKs), the loss of cyclin D1, the upregulation of p27 kip1 , and G 1 phase cell cycle arrest (22, 62). Conversely, mechanical tension stimulates focal adhesion kinase (FAK) phosphorylation (69, 81), which can induce cyclin D1 and downregulate p21 cip1 (84). These kinds of data have lent support to the idea that cell shape-dependent G 1 phase cell cycle progression is mediated, at least in part, by stress fibers and the generation of isometric tension. However, cultur...