Calcification of porcine bioprosthetic heart valves in the subcutaneous rat model has been shown to occur predominantly within the center of the cusp (the spongiosa). To investigate whether the calcification process can be affected by structure or location, the fibrosa and ventricularis were separated and their propensity to calcify in relation to cusps that were implanted intact was examined. Porcine aortic valve cusps were fixed in 0.5% glutaraldehyde solution for 24 hours. Ten cusps were microdissected to separate the fibrosa from the ventricularis. These layers and 10 intact cusps were separately implanted into 21 day old, male Sprague-Dawley rats. The tissues were explanted at 1, 2, 3, and 4 weeks post implantation. Histologic sections were cut and prepared with Von Kossa's stain to detect phosphates and carbonates. The separated layers displayed intrinsic calcification in a pattern similar to that of intact specimens. The similarity in the pattern of both the separated layers and the intact cusps suggests that differences in collagen fiber density in the spongiosa, ventricularis, and fibrosa do not affect the calcification process. Furthermore, no calcification was observed to occur on the outer surface of the separate layers, including areas that contained residual spongiosa. This study suggests that the process of calcification remains intrinsic regardless of the structure and density of collagen fibers.