Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 microM blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of alpha-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths.