High-frequency ultrasound (HFU) offers a means of investigating biological tissue at the microscopic level. High-frequency, quantitativeultrasound (QUS) methods were developed to characterize freshly-dissected lymph nodes of cancer patients. Three-dimensional (3D) ultrasound data were acquired from lymph nodes using a 25.6-MHz center-frequency transducer. Each node was inked prior to 3D histological fixation to recover orientation after sectioning. Backscattered echo signals were processed to yield two QUS estimates associated with tissue microstructure: scatterer size and acoustic concentration. The QUS estimates were computed following established methods using a Gaussian scattering model. Four lymph nodes from a patient with stage-3 colon cancer were evaluated as an illustrative case. QUS images were generated for this patient by expressing QUS estimates as color-encoded pixels and overlaying them on conventional gray-scale B-mode images. The single metastatic node had an average scatterer size that was significantly larger than the average scatterer size of the other nodes, and the statistics of both QUS estimates in the metastatic node showed greater variance than the statistics of the other nodes. Results indicate that the methods may provide a useful means of identifying small metastatic foci in dissected lymph nodes that might not be detectable using current standard pathology procedures.