The effectiveness of prostate-specific antigen (PSA) testing for opportunistic screening of prostate cancer is not yet well established, although this practice seems to be widely accepted. To estimate the opportunistic use of PSA by general practitioners (GPs) in Italy, we conducted a survey by querying the Health Search (HS) database, which collects clinical records from a representative sample of the Italian population. Clinical computerized data from 320 GPs and 221,557 male patients were analysed. Among total requests for PSA, 3% were linked to prostate cancer, 18% to a urological disease or symptom and 79% were not linked to any urological condition (opportunistic screening). Opportunistic screening was used for 31.4% of subjects over 50 with a significant (P<0.0001) difference between geographical areas (36.4% north, 33.5% central regions, 22.9% south and isles). This distribution was very similar to prostate cancer prevalence as it resulted from the HS data. PSA testing practice showed an increase over the years (12.7% of men over 50 in 2000, 14.3% in 2001 and 15.9% in 2002). In conclusion, the practice of opportunistic screening of prostate cancer seems to be extensively adopted by Italian GPs and is becoming more and more popular, although to date it is not supported by strong scientific evidence.