Calcification of bioprosthetic heart valves is a major factor limiting their long-term function. Current methods of microscopic examination of calcific deposits require dehydration and processing of the leaflet material, e.g. wax embedding, sectioning, gold coating. Confocal laser scanning microscopy is a new technique which allows serial optical sectioning of thick biological specimens in their normal hydrated state. The current study has examined bovine pericardium and porcine aortic valve materials calcified in vitro under static and dynamic conditions. A series of clinical explants of bovine pericardial and porcine aortic valve types has also been examined. The calcium-specific stain, Alizarin Red S, has been used as a fluorescent marker for calcium deposits. Fluorescent images, generated by Argon ion laser light at 488nm, have been obtained at varying depths into samples. These have been reconstructed to demonstrate the relationship between calcium deposits and autofluorescent collagen fibers. The patterns of calcification were similar in both in vitro and explant valve material. The calcification was of three main patterns: calcium depositing longitudinally on collagen fiber bundles; calcium forming a banded pattern perpendicular to the fiber direction and large, dense, focal deposits obliterating the underlying collagen structure. Involvement of collagen fibers in the calcification pattern was a consistent finding in all sample types. The method is a useful addition to the tools available for study of calcification processes. The potential exists for three-dimensional reconstruction of leaflet architecture and its inter-relationship with calcium deposition, in a normally hydrated state.