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This paper describes a fabrication technique for building three-dimensional (3-D) micro-channels in polydimethylsiloxane (PDMS) elastomer. The process allows for the stacking of many thin (less than 100-/spl mu/m thick) patterned PDMS layers to realize complex 3-D channel paths. The master for each layer is formed on a silicon wafer using an epoxy-based photoresist (SU 8). PDMS is cast against the master producing molded layers containing channels and openings. To realize thin layers with openings, a sandwich molding configuration was developed that allows precise control of the PDMS thickness. The master wafer is clamped within a sandwich that includes flat aluminum plates, a flexible polyester film layer, a rigid Pyrex wafer, and a rubber sheet. A parametric study is performed on PDMS surface activation in a reactive-ion-etching system and the subsequent methanol treatment for bonding and aligning very thin individual components to a substrate. Low RF power and short treatment times are better than high RF power and long treatment times, respectively, for instant bonding. Layer-to-layer alignment of less then 15 /spl mu/m is achieved with manual alignment techniques that utilize surface tension driven self-alignment methods. A coring procedure is used to realize off-chip fluidic connections via the bottom PDMS layer, allowing the top layer to remain smooth and flat for complete optical access.