In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 mum in diameter) were printed onto amino-silane-and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 mum diameter collagen spots, were micropatterned with a highdensity array of 30 mum x 30 mum poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.