Summary In this project we will develop a technology called electrokinetic lithography (EKL) that will fill a technology void in the current state-of-the-art 3D aligned collagen fiber microengineering techniques. To mimic the structurally heterogenous environment found in the native extracellular matrix (ECM), we will sequentially combine extensional fluid flows with electric field driven bead motion (electrokinetics) to “write” cellular-scale discontinuities between domains of aligned collagen fibers within biomimetic 3D collagen gel. Technology development will be carried out with the followings aims: 1) Establish flow-based collagen fiber alignment and characterize electrokinetic transport parameters, and 2) Develop a microfluidic platform to engineer discontinuities within aligned 3D gel environments and validate cell motility responses. The success of this project will establish a transferrable lab prototype and support unprecedented studies that explore how cells respond to local disruptions in the aligned fibrous microarchitecture. Our technique will support new lines of exploration related to motility, sensing, and cell-cell communication within structurally heterogeneous environments, and address research questions cannot be currently answered with state-of- the-art collagen alignment techniques. This project directly aligns with the NIGMS mission of developing tools that enable potentially transformative biomedical research.