OBJECTIVE This histomorphometric study compared the efficacy of a new bone graft substitute (N-HA) derived from hen eggshell, consisted of submicron scale porous hydroxyapatite structure, in the healing of 8 mm diameter critical size defects in rat calvaria. We compared N-HA alone or in combination with calcium sulfate (CS), with a commercial bone substitute, anorganic bovine bone (Bio-Oss, BO). METHODS Critical size defects were created in calvaria of 56 adult Sprague-Dawley rats. Animals were divided into four groups and treated with (1) unfilled defects, (2) N-HA grafts, (3) BO grafts and (4) N-HA/CS grafts. The percentage of new bone formed (NB%) was evaluated histomorphometrically after 6 and 12 weeks. RESULTS The N-HA group exhibited more new bone formation compared with other groups at 6 and 12 weeks. Histomorphometric analysis showed greater NB% in N-HA group (11.2% at 4 weeks and 19.2% at 12 weeks) compared with those in unfilled (3.9% at 6 weeks and 6.4% at 12 weeks), BO-treated (6.4% at 6 weeks and 8.2% at 12 weeks) and N-HA/CS-treated (6.3% at 6 weeks and 12.6% at 12 weeks) groups. The N-HA group showed significant differences in NB% compared with unfilled group at 6 weeks (P=0.016), unfilled and BO-treated groups at 12 weeks (P=0.001). The addition of CS did not enhance the NB% compared with defects grafted with N-HA alone. CONCLUSIONS N-HA was an osteoconductive bone substitute for treating osseous defects in critical size defects of rat calvaria.