Purpose: The purpose of this study was to compare the osteoblastic and osteogenic responses to titanium surfaces roughened by 1-step and 2-step acid etching. Materials and Methods: Titanium surfaces created by 1-step (AE1) and 2-step (AE2) acid-etching processes were analyzed using scanning electron microscopy (SEM), atomic force microscopy (AFM), and an optical interferometry (OI). Rat bone marrow-derived osteoblastic cells were cultured on these 2 surfaces. Cell proliferation was evaluated by counting the cells, while gene expression was analyzed using a reverse transcriptase-polymerize chain reaction. The biomechanical establishment of osseointegration was assessed via an in vivo implant push-in test in rat femurs. Additionally, the 2 surfaces were evaluated for their mechanical interlocking capability by a push-out test of titanium rods embedded in a resin block. Results: AFM analysis on a small scale of 5 µm 3 5 µm showed that the 2 surfaces were similar in topography, having comparable micron-level roughness. However, larger scale (1000 µm 3 1000 µm) SEM and OI analyses revealed that the AE1 surface consisted of supramicron convexity structures ranging from 10 µm to 50 µm in size, while the AE2 was relatively flat. No differences were found between the 2 surfaces in regard to the number of the cells proliferated or the expression of the bone-related genes. The biomechanical fixation of implants at week 2 was 22.2 ± 10.94 N and 25.4 ± 4.56 N for AE1 and AE2, respectively, with no significant difference between the 2 groups. The in vitro push-out values were 26.8 ± 7.85 N and 25.4 ± 8.56 N for AE1 and AE2, respectively, with no significant difference between the 2 groups. Conclusion: The different acid-etching procedures of titanium created similar micron-scale roughness profiles but distinct supramicron roughness characteristics. Osteoblastic function and in vivo osseointegration capacity, however, were not affected by this difference between the surfaces. Keywords: bone-titanium integration, dental implants, osteoconduction, push-in test, surface topography