The structural development of the phreno-esophageal membrane was studied in 34 human fetuses ranging from 9-26 weeks of age. Horizontal and coronal sections of the lower esophagus, cardia, and diaphragm were stained with Victoria Blue and Gomori's aldehyde fuchsin to show elastic fibers, and with peracetic-aldehyde fuchsin to show pre-elastic, oxytalan, and elastic fibers, being the elastic elements. The membrane was found to be present between the lower esophagus and diaphragm at 9 weeks. The peracetic-aldehyde fuchsin stain showed many elastic elements in this membrane, but the elastic stain showed only a few elastic fibers under 13 weeks. There was no basic structural change of the membrane from 9 to 26 weeks except for an increasing number of fibers and no basic difference from the anatomical arrangement described in the adult with fibers running from the fascial covering of the undersurface of the diaphragm upwards to the esophageal adventitia. Three dimensional observation revealed that the abdominal part of the esophagus was fixed caudally by the posterior part of the membrane. These findings suggest the position-stabilizing function of this membrane and the developmental co-operation between the hiatal crus and the lower esophageal sphincter as an anti-reflux mechanism.