Using a 20 MHz endoscopic ultrasound system, delineation of the gastric muscularis mucosae and estimation of the depth of malignant invasion was attempted by in vivo scanning during the process of routine endoscopic observation or in vitro scanning of excised sections of 34 early gastric cancers in 32 patients. The muscularis mucosae was visualized as a single hypoechoic layer in 16 of 32 lesions (50%) scanned in vitro. Comparison of lesions in which delineation of the muscularis mucosae was or was not possible revealed no significant differences with respect to either the thickness of the lamina propria and muscularis mucosae or with respect to the degree of inflammatory cell infiltration of the lamina propria or the conditions of the boundary between the lamina propria and the muscularis mucosae. This indicates that improvement of the operability characteristics of the ultrasonic apparatus will be needed to achieve improved delineation of the muscularis mucosae. The accuracy of invasion depth estimation of early gastric cancer was 67% (16 of 24 lesions scanned in vivo) and 73% (8 of 11 lesions) in cases in vivo where the muscularis mucosae and the tumor were delineated on the same screen. The principal factors causing erroneous staging were the presence of dilated benign glandular ducts, ulcer scars, and attenuation of the ultrasound waves.