In order to calculate the incidence of hepatitis C virus (HCV) antibodies in blood donors and to verify the efficacy of the pre-donation method (testing blood samples of potential donors at least 3 months in advance of their first donation), we performed a retrospective cohort study in 1995 and in the period 1996-2000 at the blood transfusion unit in Latina (Italy). Third generation ELISA method as well as RIBA-3 were used. 5,978 donors in 1995 and 20,741 in the 1996-2000 period were analysed. The age of donors (prevalently male, 78.5%) was between 18 and 65 (median 38). A total of 110 borderline and 62 positives were found with ELISA, of which 75 borderlines and 42 positives in 1995, and the remaining in the 1996-2000 period (p > 0.5). Percentage of HCV antibodies dropped from 8.5% in 1995 to 5.45% in the 1996-2000 period. The non reactivity and undetermined status were found in 82.1 and 9.4% respectively in 1995, and 69.1 and 25.45% respectively in the 1996-2000 period. The incidence of HCV antibodies was 167.28 cases per 100,000 person-years in 1995 and 4.13 per 100,000 person-years in the 1996-2000 period, 2.5 times lower than the national one. There was a statistically significant association between dental care and RIBA positivity (RR: 2.63; p = 0.045). This study, moreover, evidences how pre-donation practices, if extended to a national basis, may be able to nullify the dangers associated with post-transfusional hepatitis and to reduce the number of false positives and of blood packs donated but either unused or destroyed.