We investigated the early events of bone matrix formation, and specifically the role of fibronectin (FN) in the initial osteoblast interaction and the subsequent organization of a provisional FN matrix on different rough titanium (Ti) surfaces. Fluorescein isothiocyanate-labelled FN was preadsorbed on these surfaces and studied for its three-dimensional (3-D) organization by confocal microscopy, while its amount was quantified after NaOH extraction. An irregular pattern of adsorption with a higher amount of protein on topographic peaks than on valleys was observed and attributed to the physicochemical heterogeneity of the rough Ti surfaces. MG63 osteoblast-like cells were further cultured on FN-preadsorbed Ti surfaces and an improved initial cellular interaction was observed with increasing roughness. 3-D reconstruction of the immunofluorescence images after 4 days of incubation revealed that osteoblasts deposit FN fibrils in a specific facet-like pattern that is organized within the secreted total matrix overlying the top of the samples. The thickness of this FN layer increased when the roughness of the underlying topography was increased, but not by more than half of the total maximum peak-to-valley distance, as demonstrated with images showing simultaneous reconstruction of fluorescence and topography after 7 days of cell culture.