OBJECTIVE To determine the value of a hypoechoic lesion (HL)-directed biopsy in addition to a systematic sextant biopsy for detecting prostate cancer. SUBJECTS AND METHODS Within the European Randomized study of Screening for Prostate Cancer, 37 627 assays for prostate-specific antigen (PSA) were done in men aged 55-75 years (screening round 1-3, interval 4 years). A PSA level of >= 3.0 ng/mL prompted a systematic transrectal ultrasonography (TRUS)-guided lateralized sextant biopsy (4986 biopsy sessions were evaluated). If there was a HL, an additional lesion-directed biopsy was taken. RESULTS At the initial screening, 1840 men were biopsied and 532 cancers were detected (28.9%). Of the men biopsied, 436 had a HL and an additional biopsy (23.7%). In these men, 230 cancers were detected (52.8%). In 3.5% (eight of 230) only the HL-directed core showed malignancy. At the repeat and third screening, respectively, 19.3% and 18.9% of the men biopsied had prostate cancer, 16.8% and 9.3% had an HL and the additional core detected two (2.2%) and one (5.9%) cancers. At the first screen most cancers found by the additional core were clinically relevant. In later screens these cancers seemed to be minimal. CONCLUSIONS The performance of TRUS as a screening tool is poor. The value of the additional core was limited as only 3.5% of the visible cancers were detected solely by the additional biopsy (round 1). However, a substantial part of these cancers were clinically relevant and would have been missed without the additional biopsy. This finding was less clear in screening round 2 and 3, even in men who were not previously biopsied.