The topic of this study is to obtain the constitutive equations for two layers in the oesophagus from inflation experiment of each layer in the in vivo state and to calculate the corresponding stress distribution referenced to a new stress-free state for multi-layered tissue. The oesophagus is treated as a two-layered structure consisting of an inner submucosa layer and an outer muscle layer. An anisotropic exponential pseudo-strain energy density function is used as the constitutive equation to fit the experimental loading curve and for the calculation of the stress distribution in each layer. Significant differences in the constitutive parameters and zero-stress parameter were found between the submucosa layer and the muscle layer. The stress in the submucosa layer was higher than that in the muscle layer and intact (non-separated) wall under the same loading conditions. The submucosa layer was stiffer than the muscle layer and the intact wall. The zero-stress state of the intact oesophagus and the zero-stress state after separation were used as a reference to compare the stress-strain distributions. The residual strain was discontinuous when using the zero-stress state after separation as a reference. Furthermore, the circumferential stress when using the zero-stress state of the intact wall as a reference was about 100 times higher than that referenced to the zero-stress state after separation. Hence, it is important to use the zero-stress state obtained after the layer separation as the stress-free state in the study of multi-layered tissue. (C) 2003 IPEM. Published by Elsevier Ltd. All rights reserved.