We describe the results of a study to evaluate the intra- and inter-observer variability and reliability of prostate volume measurements made from transrectal ultrasound (TRUS) images, using either the (optimal) height-width-length (HWL) method (V = pi/6 HWL) with two-dimensional (2D) TRUS images (obtained as cross-sections of three-dimensional [3D] TRUS images) or manual planimetry of 3D TRUS images (the 3D US method). In this study, eight observers measured 15 prostate images, twice via each method, and an analysis of variance (ANOVA) was performed. This analysis shows that, with the 3D US method, intra-observer prostate volume estimates have 5.1% variability and 99% reliability, and inter-observer estimates have 11.4% variability and 96% reliability. With the HWL method, intra-observer estimates have 15.5% variability and 93% reliability, and inter-observer estimates have 21.9% variability and 87% reliability. Thus, in vivo prostate volume estimates from manual planimetry of 3D TRUS images have much lower variability and higher reliability than HWL estimates from 2D TRUS images.