Quantitative imaging methods using high-frequency ultrasound (HFU, 20 MHz)offer a means of characterizing biological tissue at the microscopic level. Inthis study, three-dimensional (3D) quantitative-ultrasound (QUS) methods weredeveloped to detect metastases in freshly-dissected lymph nodes of cancerpatients. 3D ultrasound radio-frequency data were acquired using a 26-MHzcenter-frequency transducer, and each node was inked prior to tissue fixation torecover orientation after sectioning for 3D histological evaluation.Backscattered echo signals were processed using 3D cylindricalregions-of-interest (ROIs) to yield eight QUS estimates associated with tissuemicrostructure. The first four QUS estimates (i.e., effective scatterer size,acoustic concentration, intercept, and slope) were estimated from a normalizedbackscattered spectrum, and the other four QUS estimates were obtained byparameterizing the envelope statistics of the ROIs using Nakagami andhomodyned-K models. These QUS methods were applied to 145 lymph nodes from 95colorectal and gastric cancer patients. The ability of these eight QUS estimatesto classify lymph nodes and detect cancer was evaluated using ROC curves. Anarea under the ROC curve of 0.971 with specificity and sensitivity of 91% (usinga leave-one-out procedure) were obtained by combining effective scatterer sizeand one envelope parameter based on the homodyned-K distribution. Therefore,these advanced 3D QUS methods potentially can be valuable for detecting smallmetastatic foci in dissected lymph nodes.