We reported the use of high-frequency ultrasound biomicroscopy (UBM) in the quantitative analysis of early tumor growth in mice bearing melanoma xenografts in a noninvasive longitudinal assay. Initially, measurements of tumor width, depth and length were obtained using on-screen UBM calipers in real time and tumor volume was calculated with the standard ellipsoid formula w d l pi/6. We were able to detect initiating minute tumor nodules, with the lower limit of detection at approximately 0.01 mm(3) in volume. Successive parallel cross-sectional UBM images (33 microm step) encompassing the complete length of these tumors were also obtained and reconstructed into 3-D representations. Subsequent segmentational volumetric analysis provided a measure of tumor volume. Volume measurements using the two techniques were highly correlated when all 33 xenografts were studied (r = 0.9813, p < 0.0001) and a lower degree of correlation was measured with a subset of early small tumors (r = 0.7973, n = 16, p = 0.0004). Further analysis demonstrated that 3-D segmentational volumetric analysis yielded volume estimates that were often smaller than the caliper-and-formula calculation for most early developing xenografts. Thus, 3-D UBM imaging and segmentation is expected to be especially valuable for small tumors that were observed to grow in irregular shapes other than ellipsoids.