A new technique to fabricate 3D microchannels using polydimethylsiloxane (PDMS) elastomer material is presented. The process allows for the stacking of many thin (about 100 micrometers thick) patterned PDMS layers to realize complex 3D channel paths. Replica molding method is utilized to generate each layer. The master for each layer is formed on a silicon wafer using SU-8 positive relief photoresist. 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 (RIE) 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 than 15 micrometers 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. After fabricating 3D channel paths, the hydrophobic surfaces of the inside channel walls can be activated (hydrophobic to hydrophilic) an oxygen plasma RIE system.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.