Cell function on biomaterials may depend on surface chemistry and concentration (as well as conformation) of protein molecules. To understand the interplay between these two effects, fibronectin (Fn) was physi-sorbed on a smooth, activated poly(dimethylsiloxane), films spun cast on silicon wafers. Contact angle goniometry, ellipsometry, Atomic force microscopy and Rutherford backscattering spectrometry were used to characterize the nanoscale roughness and thickness of the films. The films were activated by exposure to 30 min ultraviolet ozone radiation. Water contact angle measurements indicated higher hydrophobicity (> 100°) prior to surface activation. Tapping mode AFM scans showed that the activation process produced a rougher substrate (Ra > 0.50 nm). Fibronectin surface coverage after incubating PDMS in 2.5μg/mL of Fn was significantly higher than on non-activated surface, possibly due to favorable hydrophobic interactions between PDMS and Fn. To investigate the effect of surface activation on MC3T3-E1 osteoblast-like cells, cell spreading on PDMS and activated PDMS (30 min) coated with 2.5 μg/mL Fn was studied. Cells plated on the activated Fn-coated PDMS, for 15 min, in DMEM (with serum) showed higher cell attachment. Cell spreading after 72 h plating was clearly favored on the hydrophilic substrates as well. The increase in cell area is attributed to favorable conformational changes in absorbed Fn molecules on these substrates. © 2004 Materials Research Society.