Although several reports concerning the detection of hepatitis C virus (HCV) by in situ hybridization have been published, there are no data concerning the relative viral load in infected hepatocytes or about its relation with serum viremia levels. To address these issues, liver biopsies from 10 patients with chronic hepatitis C were analyzed by in situ hybridization and digital image analysis of hybridization signals. Serum HCV RNA levels were measured using the Amplicor Monitor test. HCV RNA was detected by in situ hybridization in the hepatocytes of the ten liver samples. The hybridization signals were mainly found in the cytoplasm. The relative viral load per infected cell fit the second order polynomial curves in all cases. The minimum and maximum relative viral load per infected hepatocyte differed in the ten cases; however, large differences were not observed in the mean relative viral load among the samples, especially when compared with the increasing values detected for copy number per milliliter in serum. The percentage of infected cells ranged from 4.8% to 87.6% in the ten cases. The percentage of positive cells correlated with the serum viremia levels. Our data suggest that HCV viremia does not depend on the relative viral load per infected cell but on the number of infected hepatocytes.