The authors used rapid prototyping to produce three-dimensional hydroxylapatite scaffolds with controlled, fully interconnected porosity. The purpose of this study was to illuminate the effect of hormones on the osteogenic differentiation and to investigate how osteoblasts colonize the three-dimensional scaffold focusing on the formation of the cellular network. Preosteoblasts were seeded onto scaffolds, were optionally treated with the osteogenic hormones triiodo-L-thyronine (T3) and 1,25-dihydroxyvitamin-D3 (D3), and the expression of osteoblastic marker genes was investigated. Confocal laser scanning microscopy was used to investigate the three-dimensional growth behavior. Culturing cells on scaffolds strongly increased the expression of osteocalcin, osteoprotegerin, Runx2, and receptor activator of NFkB-ligand (RANKL). Treatment with T3 increased the expression of osteocalcin but did not change that of osteoprotegerin and Runx2. Treatment with D3 inhibited the expression of osteocalcin, Runx2, and osteoprotegerin, Both hormones had similar effects in the three-dimensional system as found in two-dimensional cultures although more accentuated, indicating that preosteoblasts behave more naturally on three-dimensional structures. The osteoblasts colonized the three-dimensional squared pores of scaffolds by form-ing a cellular network with a round central channel keeping it into the depth and depositing collagen fibrils. These results provide insight how osteoblasts colonize a three-dimensional system and underline the importance of this environment in osteoblastic differentiation studies. © 2006 Wiley Periodicals, Inc.