Aims Hepatitis C virus antigen (HCV-Ag) detection requires retesting for samples with grey zone results (GzR), adding cost and time and decreasing reliability. Our aim in this study was to evaluate the frequency and significance of GzR during the use of the automated Architect HCV-Ag assay in routine clinical practice. Methods and Results We studied HCV-Ag levels in 952 serum samples using the ARCHITECT HCV-Ag assay. GzR were detected in 33 samples; 25 were reactive on retesting and 19 were anti-HCV positive. Seventeen of these 19 samples were tested for HCV-RNA and were all reactive (viral loads <10(4) IU ml(-1)). The remaining six samples were anti-HCV nonreactive and had undetectable HCV-RNA. Eight GzR samples were nonreactive on retesting, seven were anti-HCV nonreactive (three underwent HCV-RNA quantification and were all nonreactive), and one was anti-HCV reactive (HCV-RNA nonreactive). No significant differences were found on comparing HCV-Ag values. Conclusions Grey zone results found to be negative on retesting do not need additional technique testing, except in donor screening scenarios, where the use of molecular methods would be advisable. Significance and Impact of the Study The proposed diagnostic algorithm confirms that, eventhough GzR occur, hepatitis C virus antigen is a robust alternative to HCV-RNA detection in the active detection of infections.