The risk of transmitting cell-associated human immunodeficiency viruses (HIV- 1/-2), human T cell lymphotropic viruses (HTLV-I/-II), and cytomegaloviruses (CMV), and of transmitting cell-free HIV and hepatitis B and C viruses (HBV and HCV) from fully-screened blood can be reduced by in vitro inactivation/removal (IVIR) of blood-borne viruses (BBV). We propose a three-step cascade process for IVIR of BBV carried out on whole blood during routine blood processing: (1) selective removal of white blood cells, which harbor and permit replication of cell-associated BBV; (2) receptor-mediated removal of the cell-free BBV from leukocyte-free whole blood; and (3) biochemical or chemical inactivation of residual BBV by adding pharmacologically-acceptable agents that are biocompatible with blood. For testing this concept, our experimental design employs HIV as a prototype BBV, because HIV occurs both in cell-associated and cell-free forms and can be monitored quantitatively by complementary in vitro amplifications, biochemically by the polymerase chain reaction (PCR) and biologically by co-cultivation with CD4-positive cells. Because unacceptable platelet loss occurs during leukocyte depletion of whole blood, two different polyester filters are currently used for packed red cells and platelet concentrates. The addition of biotinylated receptor proteins (cD4-CD26) or human antibodies to HIV envelope proteins, bound to streptavidin-coated magnetic beads, will enable specific capture of both cell-free HIV and HIV-immune-complexes from the leukocyte-filtered blood. We will investigate multi-target ribozymes for enzymatic inactivation of HIV RNA and/or ascorbic acid for chemical inactivation of residual virions. Results of the studies with HIV will provide an applicable paradigm for analogous IVIR of cell-free HBV and HCV. Although we do not expect significant changes in membrane structure and function of red cells and platelets, we will perform hematological investigations to validate the safety and effectiveness of blood treatment for IVIR of BBV. Ultimately, our research and development efforts could culminate in a collaboration with manufacturer(s) capable of providing an integrated system suitable for routine IVIR of BBV in transfusion practice.