Materials with spatially resolved surface chemistries (i.e., patterned surfaces) were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, protein synthesis, cytoskeletal organization, gene expression). Surfaces were fabricated using photolithographic and photoinitiation polymerization, resulting in islands of cell-binding N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDS) separated by a non-adhesive interpenetrating polymer network of poly(acrylamide-co-ethylene glycol) (P(AAm-co-EG)). Surfaces were fabricated with over 3,800 adhesive islands/cm², allowing for isolation of single cells with projected areas ranging from 75 μm² to 10,000 μm². Cytoskeletal staining of cells cultured on these multi-domain patterned surfaces revealed that cells on islands < 400 μm² did not form stress fibers, while cells on larger EDS islands organized their F-actin filaments preferentially at the periphery of the cell and also at high stress points (e.g., comers of square islands). Subsequent experiments were performed on patterned surfaces with only one EDS adhesive island geometry and projected area (400, 1600, 3600,6400, or 10,000 μm²). Surfaces were examined to determine the influences of island projected area on cell distribution (the percentage of islands occupied by one cell), and cell projected area on proliferation and collagen expression rates. Proliferation rates were found to increase as EDS island projected area increased but all projected areas exhibited a decrease to minimal levels (<5% cell dividing) by 5 days. Type I collagen synthesis was determined strongly coupled to cell projected area, with a significantly higher percentage of cells synthesizing collagen when the island projected area was ≥ 1600 (μm² compared to 400 μm² EDS islands or control homogeneous EDS surfaces. Finally, the time required for a bone-derived cell population to initiate osteocalcin expression, an osteoblast specific marker, was determined using reverse transcription (RT) in situ polymerase chain reaction (PCR). Osteocalcin expression was observed after only 4 days in vitro from bone-derived cells maintained on patterned surfaces. In conclusion, these surfaces provide a useful tool for researching the influences of cell morphology and cytoskeletal organization on cell function.