Magnetic resonance imaging (MRI)-targeted, 3D transrectal ultrasound (TRUS)-guided “fusion” prostate biopsy aims to reduce the 21–47% false negative rate of clinical 2D TRUS-guided sextant biopsy. Although it has been reported to double the positive yield, MRI-targeted biopsy still has a substantial false negative rate. Therefore, we propose optimization of biopsy targeting to meet the clinician’s desired tumor sampling probability, optimizing needle targets within each tumor and accounting for uncertainties due to guidance system errors, image registration errors, and irregular tumor shapes. As a step toward this optimization, we obtained multiparametric MRI (mpMRI) and 3D TRUS images from 49 patients. A radiologist and radiology resident contoured 81 suspicious regions, yielding 3D surfaces that were registered to 3D TRUS. We estimated the probability, P, of obtaining a tumor sample with a single biopsy, and investigated the effects of systematic errors and anisotropy on P. Our experiments indicated that a biopsy system’s lateral and elevational errors have a much greater effect on sampling probabilities, relative to its axial error. We have also determined that for a system with RMS error of 3.5 mm, tumors of volume 1.9 cm3 and smaller may require more than one biopsy core to ensure 95% probability of a sample with 50% core involvement, and tumors 1.0 cm3 and smaller may require more than two cores.