To understand the physiology and pathology of the esophagus, it is necessary to know the mechanical properties and their dependence on the structural components. The aim of this study was to investigate the effect of collagenase and elastase on the morphological and biomechanical properties in the no-load and zero-stress states in the rat esophagus. Twenty tissue rings from each esophagus of seven normal rats were sectioned in an organ bath containing calcium-free Krebs solution with dextran and EGTA. After the rings were photographed in the no-load state, 8 of the 20 rings were separated into mucosa-submucosa and muscle rings and the rings were transferred to four different solutions containing collagenase, elastase, or corresponding control solutions. The rings were cut radially to obtain the zero-stress state and photographed again. The thickness, area, and opening angle were measured from the digitized images. The collagen and elastin area fractions were determined from histological slides with Van Gieson and Weigert's elastic stain. The opening angles and residual strain did not differ in the nonseparated enzyme-treated rings and control rings. However, in the separated mucosa-submucosa ring the opening angle was significantly smaller after treatment than that in control rings (P < 0.01). Collagenase and elastase reduced collagen and elastin in the mucosa-submucosa layer about 40% in the nonseparated wall and 54% in the separated mucosa-submucosa layer (P < 0.01). Collagenase and elastase increased the thickness in the separated mucosa-submucosa layer compared to the control (P < 0.05). Disconnection between the epithelia and the lamina propria was histologically observed after elastase digestion. In conclusion, collagenase and elastase caused the opening angle and the residual strain in the separated mucosa-submucosa layer to decrease. The opening angle of the separated mucosa-submucosa layer depended to some extent on the fraction of collagen and elastin.