We have reported that vasoactive agonists induce coordinated changes in stiffness and integrin-mediated cell adhesion in vascular smooth muscle cells (VSMCs), i.e., vasoconstrictors cause coordinated increases in VSMCs stiffness and adhesion and vasodilators cause opposite results[1]. In these studies a temporal analysis was performed to examine the dynamic effects of vasoactive agonists on VSMCs stiffness and cortical cytoskeletal structure. Atomic force microscopy (AFM) was used to perform real-time measurements of cell stiffness using a nanoindentation protocol (0.1 Hz). Data were analyzed by spectral analysis (Eigen decomposition) and[2]. VSMC stiffness was found to exhibit temporal oscillations with three distinct frequency components at 0.001/1.0, 0.004/0.4, and 0.07/0.3 Hz/kPa frequency/amplitude, respectively. Following treatment with angiotensin II (ANG II, 10-6M), amplitude of the oscillations increased significantly (+100%), whereas adenosine (ADO, 10-4M), a vasodilator, reduced oscillation amplitudes (-30%). To examine whether ANG II and ADO also altered cortical cytoskeletal structure as part of their action, the VSMC membrane topography of the sub-membranous actin cytoskeleton (100-300 nm depth) was acquired by contact mode imaging with AFM. Height and deflection signals from the topographical images were analyzed (Matlab) to compare the cortical actin fiber distribution and orientation before and after treatment with ANG II or ADO (20 min). These data show that ANG II increased the parallel alignment, density and thickness of cortical stress fibers, consistent with a rapid remodeling process involving increased actin polymerization. By comparison, ADO decreased stress fiber alignment, density and thickness of the stress fibers, consistent with disassembly of actin stress fibers. These findings demonstrate that vasoactive agonists dynamically alter temporal behavior and cytoskeletal structure of the VSMCs. These events appear to play an important role in the contractile behavior of VSMCs to vasoactive agonists.