Morphological alterations of the liver from rhesus macaque (Macaca mulatta) experimentally infected with hepatitis C virus (HCV) were analyzed using electron microscopy. The localization of viral RNA and proteins inside hepatocytes was demonstrated using in situ hybridization and immunoelectron microscopy techniques. The animals were inoculated by different routes. The infection was successful only by use of the intrasplenic approach to HCV infected autogenic hepatocyte transplant. The inoculum used to infect the hepatocytes was characterized as genotype 3 with 107 RNA copies/mL. In situ hybridization was performed using a complementary negative and positive strand probe made with the specific primer. Despite that the level of HCV infection was considered to be low, we were able to detect and localize viral positive and negative RNA strands and viral proteins in altered membranes of the rough endoplasmic reticulum in infected liver cells, showing evidence of viral replication in vivo.