Surveillance for hepatitis C virus (HCV) is limited by the challenge of differentiating between acute and chronic infections. In this study, we evaluate a cross-sectional testing strategy that identifies individuals with acute HCV infection and we estimate HCV incidence. Anti-HCV-negative persons from four populations with various risks, i.e., blood donors, Veterans Administration (VA) patients, young injection drug users (IDU), and older IDU, were screened for HCV RNA by minipool or individual sample nucleic acid testing (NAT). The number of detected viremic seronegative infections was combined with the duration of the preseroconversion NAT-positive window period (derived from analysis of frequent serial samples from plasma donors followed from NAT detection to seroconversion) to estimate annual HCV incidence rates. Projected incidence rates were compared to observed incidence rates. Projected HCV incidence rates per 100 person-years were 0.0042 (95% confidence interval [95% CI], 0.0025 to 0.007) for blood donors, 0.86 (95% CI, 0.02 to 0.71) for VA patients, 39.8 (95% CI, 25.9 to 53.7) for young IDU, and 53.7 (95% CI, 23.4 to 108.8) for older IDU. Projected rates were most similar to observed incidence rates for young IDU (33.4; 95% CI, 28.0 to 39.9). This study demonstrates the value of applying a cross-sectional screening strategy to detect acute HCV infections and to estimate HCV incidence.It is estimated that hepatitis C virus (HCV) infects nearly 4 million persons in the United States (3) and 130 million persons worldwide (19). In the United States, chronic HCV infection is the primary cause of end-stage liver disease resulting in liver transplantation (2). Transfusion-transmitted HCV has been virtually eliminated in the developed world as a result of routine screening of blood products by using progressively more sensitive antibodies and, since 1999, minipool nucleic acid testing (NAT) (25, 47). However, new infections still occur at high rates in other at-risk populations, especially injection drug users (IDU). Worldwide, the prevalence of HCV infection among IDU ranges from 25% to 80% (14,21,22,27,34,45), and the incidence among younger IDU ranges from 9% to 38% per year (13,16,23,37,49).Monitoring HCV infection has principally been limited to serosurveys detecting HCV-specific antibodies (anti-HCV), using enzyme immunoassays (EIAs) and confirmatory recombinant immunoblot assays (RIBAs). However, these cannot differentiate between acute, recent, chronic, and resolved infections, distinctions which would be very useful for HCV surveillance. Recent infection represents the period from exposure through early seroconversion, with acute infection comprising the viremic preseroconversion phase (the viremic preseroconversion "window period"). Although most acutely infected individuals (60 to 80%) will become chronic carriers of the virus, infection is self-limited in an average of 26% of individuals who spontaneously resolve viremia (2, 36). Very few data are available about the rate of acute HCV infection (an...