Functional biological synthetic composite (BSC) membranes were made using phospholipids, biological membrane proteins and permeable synthetic supports or membranes. Lipid bilayers were formed on porous polycarbonate (PC), polyethylene terephthalate (PETE) and poly (l-lactic acid) (PLLA) membranes and in 10-100 microm laser-drilled pores in a 96-well plastic plate as measured by increased resistance or decreased currents. Bilayers in 50 microm and smaller pores were stable for up to 4 h as measured by resistance changes or a current after gramicidin D reconstitution. Biological membrane transport reconstitution was then carried out. Using vesicles containing Kv1.5 K(+) channels, K(+) currents and decreased resistance were measured across bilayers in 50 microm pores in the plastic plate and PLLA membranes, respectively, which were inhibited by compound B, a Kv1.5 K(+) channel inhibitor. Functional reconstitution of Kv1.5 K(+) channels was successful. Incorporation of membrane proteins in functional form in stable permeable membrane-supported lipid bilayers is a simple technology to create BSC membranes that mimic biological function which is readily adaptable for high throughput screening.