To investigate whether certain handling and storage conditions of serum samples could alter the sensitivity and specificity of the hepatitis C virus (HCV) RNA assay, we studied serum samples obtained from five patients known to be positive for HCV RNA and two patients with autoimmune chronic active hepatitis. Samples were subjected to one of the following conditions: (1) immediate storage at -20 degrees C, (2) five freeze-thaw cycles, (3) storage at 4 degrees C for 5 days, and (4) storage at room temperature for 5 days. Detection of HCV RNA was performed by polymerase chain reaction. Titers of HCV RNA were determined by serial end point dilutions. We found that the titer of HCV RNA was reduced by only one logfold in samples subjected to conditions 2 through 4 in two of the five patients. False-positive results were not seen with the serum samples that were subjected to similar conditions from the two negative control patients. We conclude that serum HCV RNA is resistant to degradation under routine laboratory handling and storage conditions.