Toll-like receptors (TLR) have a critical role in innate immunity against pathogens. We investigated the cytokine response to TLR stimulation in peripheral blood cells of subjects infected with hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) in the Women Interagency HIV Study (WIHS) cohort. Interleukin (IL)-6 in response to TLR3 and TLR4 ligands such as polyinosinic-polycytidylic acid and lipopolysaccharide was significantly compromised in HCV-infected women. High spontaneous secretion of IL-6 suggested pre-existing cell activation as a factor mediating reduced responses to TLR3 and TLR4 stimulation. To a lesser extent, tumour necrosis factor-alpha and IL-1beta responses to TLR stimulation were also compromised. Monocytes, but not B cells or NK cells, were identified as the cell population spontaneously secreting cytokines and also as the cells responding to TLR stimulation. These results highlight a functional defect in antigen-presenting cells of women with HCV infection or co-infection. In women with existing HIV co-infection, decreased cytokine function of antigen-presenting cells suggests another mechanism contributing to immune dysfunction in addition to the HIV-associated CD4 defect.