Cells adapt in response to mechanical stimulation to ensure adequate tissue functioning. F-actin stress fibers provide a key element in the adaptation process. The high sensitivity and fast adaptation of the F-actin cytoskeleton to cyclic strain have been studied extensively in a 2-D environment; however, 3-D data are scarce. Our previous work showed that stress fibers organize perpendicular to cyclic stretching (stretch-avoidance) in three dimensions. However, stretch-avoidance was absent when cells populated a high density matrix. In this study our aim was to obtain more insight into the synergy between matrix density and the signaling pathways that govern stress fiber remodeling. Therefore we studied stress fiber organization in 3-D reconstituted collagen tissues (at low and high matrix density), subjected to cyclic stretch upon interference with molecular signaling pathways. In particular, the influence of the small GTPase Rho and its downstream effectors were studied. Only at low matrix density does stress fiber stretch avoidance show a stretch-magnitude-dependent response. The activity of matrix metalloproteinases (MMPs), Rho-kinase and myosin light chain kinase are essential for stress fiber reorientation. Although high matrix density restricts stress fiber reorientation, Rho activation can overcome this restriction, but only in the presence of active MMPs. Results from this study highlight a synergistic action between matrix remodeling and Rho signaling in cyclic-stretch-induced stress fiber organization in 3-D tissue. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.