Titanium (Ti) and Ti alloys are widely used as dental and orthopedic implants, but the effects of the surface characteristics of these materials, including roughness, on the response of target tissues in vivo are not well understood. The present study has therefore examined the effects of a moderately rough (sand-blasted, acid-etched; SLA) Ti surface, a highly rough (plasma-sprayed; TPS) surface, and a smooth surface (SMO) on bone cells in vitro. X-ray photoelectron spectroscopy showed that these surfaces had similar surface chemistry, while scanning electron microscopy suggested that the SLA provided a transiently less biocompatible surface, with initially less well-attached cells. SLA also delayed bone cell growth compared with SMO, whereas the TPS surface elicited the greatest increase in cell numbers. In addition, expression profiling using the ATLAS gene array showed marked differences in gene responses after 3 h of incubation; this increased further after 24 h, with TPS generating the largest number of up- and down-regulated genes compared with SLA and SMO. A number of osteoblast genes were also identified as 'roughness' genes on the basis of their similar response on SLA and TPS, compared with SMO. These findings show, for the first time, that the surface roughness of Ti has a profound effect on the profile of genes expressed by bone cells and suggest that improvements in the biological activity and possibly the clinical efficacy of these materials could be achieved by selective regulation of gene expression mediated via modification of surface roughness.