The current study characterized the in vitro surface reactions of microroughened bioactive glasses and compared osteoblast cell responses between smooth and microrough surfaces. Three different bioactive glass compositions were used and surface microroughening was obtained using a novel chemical etching method. Porous bioactive glass specimens made of sintered microspheres were immersed in simulated body fluid (SBF) or Tris solutions for 1, 6, 24, 48, or 72 h, and the formation of reaction layers was studied by means of a scanning electron microscope/energy dispersive X-ray analysis (SEM/EDXA). Cell culture studies were performed on bioactive glass disks to examine the influence of surface microroughness on the attachment and proliferation of human osteoblast-like cells (MG-63). Cell attachment was evaluated by means of microscopic counting of in situ stained cells. Cell proliferation was analyzed with a nonradioactive cell proliferation assay combined with in situ staining and laser confocal microscopy. The microroughening of the bioactive glass surface increased the rate of the silica gel layer formation during the first hours of the immersion. The formation of calcium phosphate layer was equal between control and microroughened glass surfaces. In cell cultures on bioactive glass, the microrough surface enhanced the attachment of osteoblast-like cells but did not have an effect on the proliferation rate or morphology of the cells as compared with smooth glass surface. In conclusion, microroughening significantly accelerated the early formation of surface reactions on three bioactive glasses and had a positive effect on initial cell attachment.