The interaction of different interferon (IFN)-α subtypes with different cell types was investigated using a unique monoclonal antibody (MAb), I-4-A. This MAb reacts in immunoassays equally with IFN-α2b and IFN-α4a, but does not inhibit the binding of IFN to cell receptors. 125I-labeled I-4-A reacted with IFN-α4a and IFN-α2b bound to receptors on Daudi cells. However, in a “double assay” developed using Daudi cells to measure antiviral and antiproliferative activity, I-4-A neutralized both activities of IFN-α4a, but neither of IFN-α2b. Similarly, in studies on the activation of natural killer (NK) cells, 1-4-A neutralized the effect of IFN-α4a but not that of IFN-α2b. In contrast, when cell lines other than lymphoid were studied, e.g., HEp 2 and WISH cells, I-4-A neutralized the antiviral activity of both IFN-α subtypes. The neutralization of one IFN-α subtype but not another on lymphoid cells suggests a difference either in the receptor-bound form of the subtypes, or in subsequent interactions prerequisite for activation of these cells. Furthermore, the neutralization of a particular IFN subtype, α2b, on epithelial-derived but not lymphoid cells suggests differences in the IFN-receptor complex or the mechanisms of cell activation between these cell types. An implication from these studies is that some IFN-α subtypes can exert different functions on lymphoid and epithelial cells.