"Saturation binding experiments" were carried out to characterize the attachment of reovirus to mouse L fibroblasts. Scatchard analysis of data obtained from such experiments suggests that one homogeneous set of noncooperative, high affinity binding sites are involved in reovirus attachment. It is estimated that L cells possess between 3 and 5 X 10(5) reovirus binding sites per cell and that the equilibrium dissociation constant (KD) is approximately 3 X 10(-9) M. Scatchard analysis of data from similar experiments, carried out in the presence of anti-sigma 1 and anti-sigma 3 antibodies, revealed that although both antibodies prevent viral attachment, they exhibit distinct binding inhibition characteristics: anti-sigma 1 effectively abolishes high-affinity, specific binding, whereas anti-sigma 3 apparently blocks low-affinity, nonspecific interactions. The nature of the L-cell receptor was then probed using various enzymes and reagents, and compared with that of the reovirus receptor on human type O erythrocytes. It was found that whereas reovirus hemagglutination (HA) is inhibited by pretreatment of erythrocytes with various proteases or neuraminidase, virus binding to L cells is unaffected by such treatments. Neither HA nor cell binding is inhibited by the various sugars tested, including N-acetyl-D-glucosamine, which was previously reported to inhibit reovirus HA (L. D. Gelb and A. M. Lerner, 1965, Science 147, 404-405). Both L cells and erythrocyte reovirus receptors are nevertheless highly sensitive to periodate treatment, which presumably destroys the high-affinity reovirus binding sites since protein sigma 1, which is capable of attaching to L cells by itself, does not bind to cells pretreated with periodate. It is therefore concluded that sugar residues on the receptor may be involved in this specific interaction. The possibility that gangliosides may serve as reovirus receptors was also probed. It was found that bovine brain gangliosides, but not cerebrosides, readily aggregate reovirus, inhibit HA, and block viral attachment to L cells. However, binding of protein sigma 1 to L cells is unaffected by gangliosides. Inhibition of reovirus HA and L-cell binding by these gangliosides is therefore most likely due to a steric hindrance effect brought about by interactions between the gangliosides and other components of the outer viral capsid.