Because previous reports found an association between hepatitis C virus (HCV) coinfection and progression of human immunodeficiency virus (HIV) disease, we investigated whether HIV and HCV may reciprocally influence viral replication in monocyte lineage cells in vivo. Using a novel technique called simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI), we rapidly and unequivocally identified HCV reservoirs in peripheral blood from HCV-infected individuals with and without HIV coinfection. We found that HCV infects both CD14(+), CD16(+)(+) monocytic cells and CD14(+)(+), CD16(+)(+) monocytic cells but not CD14(+)(+), CD16- cells in individuals infected with HCV with or without HIV coinfection. To address these HCV tropism differences, we found that the HCV receptor CD81 is highly expressed on CD14(+), CD16(+)(+) and CD14(+)(+), CD16(+)(+) cells but not on monocytes (CD14(+)(+), CD16-). These findings have important implications for the diagnosis and treatment of HCV infection, mother-to-child transmission of HCV, and possible virus-virus interactions in HCV-HIV coinfected individuals.