Abstract To investigate the effect of the sequential delivery of bone morphogenetic proteins BMP-2 and BMP-7 on bone regeneration in rat calvarial defects (40 Sprague-Dawley rats, 8 mm defect size), all animals were treated with a hydroxyapatite (HA)/tricalcium phosphate (TCP) bone graft covered with a collagen membrane. The experimental groups were as follows: (1) control group: unmodified collagen (no treatment); (2) BMP-2 group: 5 μg of BMP-2; (3) hep-BMP-7 group: 5 μg BMP-7 chemically bound to heparinized collagen; and (4) BMP-2/hep-BMP-7 group: 2.5 μg BMP-7 bound to heparinized collagen and subsequently treated with 2.5 μg BMP-2. Defect healing was examined at 2 and 8 weeks after surgery. The BMP-2 group showed the largest new bone area at week 2 (29.3 ± 7.3%; P = 0.009); new bone areas in the hep-BMP-7 and BMP-2/hep-BMP-7 groups were similar (11.8 ± 3.4% and 12.9 ± 5.71%, respectively; P = 0.917). After 8 weeks, the BMP-2/hep-BMP-7 group showed the largest new bone area (43.3 ± 6.2%), followed by the BMP-2 and hep-BMP-7 groups ( P = 0.013). Accordingly, in comparison with single deliveries of BMP-2 and BMP-7, sequential delivery of BMP-2 and BMP-7 using a heparinized collagen membrane significantly induced new bone formation with a smaller quantity of BMP-2 in rat calvarial defects.