Rat osteoblast cultures were maintained from 24 h to 6 weeks on hydroxyapatite (HA)- or titanium (Ti)-coated smooth and micromachined grooved substrata in medium supplemented with L-ascorbic acid-2-phosphate and beta-glycerophosphate to promote mineralization. The HA coatings, approximately 1 microm thick, were characterized using X-ray diffraction, surface roughness, and scanning electron microscopy (SEM). Osteoblasts elongated, aligned, and moved in the direction of the grooves on both Ti and HA grooved surfaces. HA surfaces produced significantly more bone-like nodules than Ti surfaces. All grooved substrata produced significantly more nodules than smooth surfaces. These results are consistent with the hypothesis that substrata can increase osteogenesis by formation of an appropriate microenvironment. There was also a statistically significant interaction between topography and chemistry in the formation of mineralized nodules. A strong correlation (r = 0.958) between alkaline phosphatase (Alk-P) at 2 weeks and nodule counts at 6 weeks was observed, suggesting that Alk-P may possibly be used as a leading indicator of osteogenesis on microfabricated surfaces. The results of this study indicate that surface topography and chemistry can affect osteogenesis, and that interactions between chemistry and topography can occur.