The present study describes a new three-dimensional (3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and beta-glycerophosphate (beta GP). Under these mineralization conditions, mRNA expressions of procollagen alpha 2(I) and [3H]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen alpha 2 (I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fibrils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.