We determined how long cryopreserved aortic, pulmonic, mitral, and tricuspid valve homografts could be stored in a deep freezer (-80 degrees C) without compromising fibroblast viability. Valves harvested from 20 anesthetized mongrel dogs were grouped into nonfrozen control, frozen and stored in liquid nitrogen (-196 degrees C), and frozen and stored in a deep freezer (-80 degrees C). Frozen groups were divided into subgroups and stored for 2, 4, 8, or 12 weeks. A leaflet of each valve was divided into three fragments, and fibroblast viability was analyzed by flow cytometry. Cell viability was defined as staining by fluorescent diacetate but not by propidium iodide. The viability of untreated control valves from all four sites was about 70%, decreasing to about 50% when treated with low doses of antibiotics. The viability of frozen valves stored in liquid nitrogen was about 45% without a significant difference among storage periods. The viability of valves frozen and stored in a deep freezer was significantly lower than for the liquid nitrogen group at 2 weeks for the mitral valve and at 4 weeks for other valves. These results suggest that homografts can be stored in a deep freezer for up to 2 weeks without deterioration.