Background: In prostate cancer (PCa) screening men and their physicians aim to rule out the presence of potentially life threatening PCa. To date, prostate specific antigen (PSA) testing and systematic prostate biopsy (Bx)-in case of an elevated PSA-are still the main modes of PCa detection. Often uncertainty remains when a PSA-test is < 3.0 ng/mL or a Bx shows a benign result, leading to the continuous repeating of procedures. Here we assess the potential consequences of false negatives by studying follow-up data of a purely PSA-based approach with applying sextant Bx, an approach considered to have a high risk of missing PCa diagnosis. Methods: Our study population consisted of 19,970 men from the ERSPC project section Rotterdam, initially screened in 1993-1999. We assessed clinically significant Gleason ≥3+4 PCa (csPCa) diagnosis within the 4-year screening interval and subsequent screening round 4 years later in men having a PSA < 3.0 ng/mL at initial screening (no Bx) and men with Bx (PSA > 3.0 ng/mL), but no PCa detected at that time. In addition, we addressed PCa mortality and PCa diagnosis for men with a negative PSA test and negative Bx, who were retested every 4 years covering a 15-year follow-up. Results: A total of 14,935 men had PSA < 3.0 ng/mL in the initial screening round, of whom 75 (0.5%) were diagnosed with csPCa at a subsequent screening examination and 2 ( < 0.1%) in the 4-year screening interval. For 2,260 men with a previously negative Bx at first screening, the figures were 17 (0.8%) and 2 (0.1%) respectively. Indolent PCa (Gleason ≥3+3) was diagnosed in 312 (2%) men with PSA < 3.0 ng/mL initially and 115 (5%) men with initial negative Bx. After a 15-year follow-up, 45 (0.3%) PCa deaths occurred in men with initially low PSA, and 29 men (0.2%) had metastasis. For men with negative Bx, 11 (0.5%) PCa deaths occurred and 4 (0.2%) experienced metastasis. Conclusions: The false negative rates for men with PSA < 3.0 ng/mL and negative sextant Bx are extremely low but not negligible. Proper risk stratification before deciding to biopsy is expected to hardly miss any clinical significant PCa diagnosis. This is especially relevant with the increased use of the relatively expensive multi-parametric magnetic resonance imaging (mpMRI) guided targeted Bx procedures.