The expression of interferon-alpha (IFN-alpha) receptors was studied on a variety of human cells, using monoiodinated IFN-alpha 2 probes. Steady-state binding at 4 degrees C revealed a single class of non-interacting IFN receptor on peripheral blood lymphocytes, and tonsillar B lymphocytes, which are both known to be G0/G1 resting cell populations. The binding affinity of this class of receptor was found to be on the order of 5 X 10(-10) M, expressed as an apparent dissociation constant (Kd). However, cells proliferating either in culture or in vivo were found to express a heterogeneity in IFN-alpha 2 binding. Such binding could be objectively resolved (by a version of the LIGAND program of P. Munson) into a two-site receptor model. Hill plots of binding to proliferating cells indicated a negative cooperativity in the interaction of IFN and receptor. The high-affinity component, expressed on proliferating cells, typically exhibits a Kd of (1-10) X 10(-11) M, while the lower-affinity component indicates a Kd of (1-10) X 10(-9) M. Furthermore, the low-affinity component is apparently expressed on the order of 10-200 times the copy number, per cell, of the high-affinity site. Affinity-labeling experiments revealed that, in addition to the 140-160-kDa IFN-binding complex reported by others, both the proliferating and non-proliferating cell populations possess a novel IFN-binding component of 60 kDa.