Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is currently employed as an autograft replacement for spinal fusion. The morphogen is incorporated onto its carrier, an absorbable collagen sponge (ACS), in the operating room. Although the effectiveness of the rhBMP-2/ACS implant in stimulating bone formation in human subjects has now been well established, further investigations of its use are necessary to deepen our understanding of its performance. The objective of the present study was to determine whether fluid released from the rhBMP-2/ACS implant could induce bone growth in tissue sites away from the implant site. We first measured the amount of protein in the fluid released from the rhBMP-2-soaked ACS during intraoperative handling. Variables included soak time and degree of compression. In the compression group that most closely approximated intraoperative conditions, more than 95% of the rhBMP-2 protein was retained by the ACS following a 15-min. soak time. This in vitro study was followed by an in vivo ectopic implant experiment using rat and rabbit models. The animal investigation compared the amount of bone induced by rhBMP-2 solution alone versus the de novo bone formation induced by rhBMP-2/ACS implants with varying concentrations of rhBMP-2. No ossicles were found at the sites where rhBMP-2 solution was injected in either animal species. Twenty-two of the 24 subcutaneous sites in the rats implanted with the rhBMP-2/ACS constructs displayed the presence of the typical 4- and 12-week ossicle. There were no noticeable differences in the size and shape of the ossicles after 4 and 12 weeks. There was a greater percentage of implant sites without ossicles in the rabbits, compared to the rats.