OBJECTIVE High-frequency ultrasound allows high-resolution imaging of anterior segment anatomy and pathology. Acoustic echo data, however, contain information relating to the microanatomic structure of the interrogated tissue which is not evident in B-mode images. The aim of this study is to develop imaging techniques to demonstrate and quantify the distribution of acoustic scattering properties in ocular tissues in three dimensions. METHODS A tumor of the iris and a hyphema were scanned using a 50-MHz ultrasound probe mounted on a computer-controlled two-axis positioning system. Scan data from sequential parallel planes were used to make three-dimensional reconstructions. Digital signal processing and a mathematical model of acoustic backscatter then were used to represent the effective size and acoustic concentration of scattering elements using a false color representation superimposed on B-mode images. RESULTS Three-dimensional reconstructions improved appreciation of the size and extent of pathology and allowed computation of tissue volumes. Parameter images demonstrated distinctive differences between diffuse and organized blood and allowed quantification of tumor scattering properties. CONCLUSIONS Three-dimensional imaging of the anterior segment with high-frequency ultrasound allows construction of perspective images, which adds to the already significant clinical use of individual high-resolution B-mode images. Acoustic backscatter properties determined by tissue microstructure can be computed from echo data and represented in false color in three-dimensional reconstructions.