BACKGROUND: Although localised prostate cancer is multifocal in most instances, the index lesion might be responsible for disease progression. OBJECTIVE: To determine the early genitourinary functional and cancer control outcomes of index lesion ablation. DESIGN, SETTING, AND PARTICIPANTS: This was a single-centre prospective development study in which 56 men were treated (July 2009-January 2011). The mean age was 63.9 yr (standard deviation 5.8) and median prostate-specific antigen (PSA) was 7.4 ng/ml (interquartile range [IQR] 5.6-9.5). There were seven (12.5%) low-risk, 47 (83.9%) intermediate-risk, and two (3.6%) high-risk cancers. INTERVENTION: Multiparametric magnetic resonance imaging (mpMRI) and prostate biopsies to localise disease, followed by index lesion ablation using high-intensity focused ultrasound. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary outcomes were genitourinary side effects measured using validated questionnaires. Secondary outcomes included absence of clinically significant disease at 12 mo. RESULTS AND LIMITATIONS: The composite of leak-free, pad-free continence, and erections sufficient for penetration decreased from a baseline frequency of 40/56 (71.4%) to 33/56 (58.9%) at 12 mo. Pad-free and leak-free, pad-free continence was preserved in 48/52 (92.3%) and 46/50 (92.0%) patients, respectively. Erections sufficient for intercourse were preserved in 30/39 (76.9%) patients. The median PSA nadir decreased to 2.4 ng/ml (IQR 1.6-4.1). At 12 mo, 42/52 (80.8%) patients had histological absence of clinically significant cancer and 85.7% (48/56) had no measurable prostate cancer (biopsy and/or mpMRI). Two (3.6%) patients had clinically significant disease in untreated areas not detected at baseline. The main study limitation is the short follow-up duration. CONCLUSIONS: Index lesion ablation had low rates of genitourinary side effects and acceptable short-term absence of clinically significant cancer. Comparative effectiveness trials are required to assess cancer control outcomes against radical therapy. PATIENT SUMMARY: In this study we looked at whether it is possible to treat the largest and highest-grade tumour in men who have more than one known prostate tumour. We show that the side effects of targeted ablation were low, with acceptable rates of early cancer control. Larger studies with longer follow-up are needed. TRIAL REGISTRATION: NCT00988130.