AbstractSuccessful bone‐tissue engineering (TE) has been reported for various strategies to combine cells with a porous scaffold. In particular, the period after seeding until implantation of the constructs may vary between hours and several weeks. Differences between these strategies can be reduced to (a) the presence of extracellular matrix, (b) the differentiation status of the cells, and (c) the presence of residual potentially immunogenic serum proteins. These parameters are investigated in two types of calcium phosphate scaffolds in a goat model of ectopic bone formation. Culture‐expanded bone‐marrow stromal cells from eight goats were seeded onto two types of hydroxyapatite granules: HA60/400 (60% porosity, 400‐μm average pore size) and HA70/800. Scaffolds seeded with cells and control scaffolds were cultured for 6 days in medium containing autologous or semisynthetic serum, in the presence or absence of dexamethasone. Other scaffolds were seeded with cells just before implantation in medium with or without serum. All conditions were implanted autologously in the paraspinal muscles. After 12 weeks, bone had formed in 87% of all TE constructs, as demonstrated by histology. Histomorphometry indicated significantly more bone in the HA70/800 scaffolds. Furthermore, a significant advantage in bone formation was found when the constructs had been cultured for 6 days. In conclusion, both scaffold characteristics (porosity) and TE strategy (culturing of the constructs) were demonstrated to be important for bone TE. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 69B: 113–120, 2004