BACKGROUND: The use of RT-PCR for diagnosis of group A rotaviruses is increasing, but up to 14% of healthy individuals may be positive by RT-PCR. If RT-PCR is not well correlated with disease, rotavirus A may not always be the cause of illness in RT-PCR positive patients with infectious intestinal disease (IID). OBJECTIVES: To describe the differences in faecal viral load between ELISA positive IID cases, RT-PCR positive cases and healthy controls. To develop a cut-off in faecal viral load for attributing illness to rotavirus A in RT-PCR positive IID cases. STUDY DESIGN: Faecal viral load was measured, using real time RT-PCR, in 118 community IID cases and 65 healthy controls, previously tested by ELISA. Cycle threshold (Ct) values from the real-time RT-PCR were used as a proxy measure of viral load. A cut-off for attributing illness to rotavirus A was selected, using ROC analysis. RESULTS: There was little overlap in viral load between ELISA positive IID cases (median Ct 17) and healthy controls (median Ct 37), but ELISA negative, RT-PCR positive IID cases (median Ct 37) had viral loads similar to healthy controls, indicating that RT-PCR is not detecting extra cases of group A rotavirus associated IID, only sub-clinical infections. The optimal cut-off in the real time RT-PCR was at Ct value 24-27. CONCLUSION: ELISA is the best method for the laboratory diagnosis of rotavirus A associated IID. If RT-PCR is used, it is advisable to use a real time platform and to use a viral load cut-off equivalent to the detection limit of ELISA.