Objectives: To evaluate the features, rates, and characteristics of prostate cancer detected during two subsequent screening rounds. Methods: Data were retrieved from the database of European Randomized Study of Screening for Prostate Cancer (ERSPC), section Rotterdam. Men, ages 55-74 yr were screened with a 4-yr interval. Different biopsy indications were used in the first and second screens in the PSA range <4.0 ng/ml. Clinical features and a total of 1548 sextant biopsies were recorded for Gleason score and tumour extent, and 550 radical prostatectomy specimens were evaluated for Gleason score, pathologic T category, and tumour volume. Results: Clinical stage, Gleason score, involvement of biopsy by tumour, and PSA levels were more favourable in patients of the second round compared with those of the first round. The number of men chosen for watchful waiting increased from 98 (10%) to 123 (22%) in the second round (p < 0.0001). In patients undergoing radical prostatectomy, median tumour volume in the first and second screening round was 0.65 and 0.45 ml (p = 0.001). Minimal cancer (cancer <0.5 ml, organ-confined, no Gleason pattern 4 or 5) was found in 122 (31.6%) in the first and 60 (42.6%) in the second screening round (p = 0.03). The 5-yr PSA progression-free survival after radical prostatectomy was 87%. Conclusions: Despite the 4-yr interval an important shift of all prognostic factors occurred in favour of round 2. In those men who underwent radical prostatectomy, 42.6% fulfilled the criteria of minimal cancer. These data suggest that overdiagnosis increases with repeat screening.