The African green monkey kidney-derived Vero cell line expresses a receptor activity for noninfectious hepatitis B surface antigen (HBsAg) particles containing the small S protein. (M.E. Peeples, K. Komai, R. Radek, and M.J. Bankowski, 1987, Virology 160, 135-142). In this report, the binding characteristics, the physiological requirements, and the functions of this receptor are further characterized. The association rate constant (ka) was determined by measuring binding during a short (10-min) incubation period to avoid the complication of dissociation. The results indicated an extremely high affinity binding: ka = 2.0 x 10(10) M-1 min-1. HBsAg particle binding to the Vero cells was also found to be slowly reversible, and dependent on temperature, pH, and Ca2+. After binding to Vero cells. HBsAg particles were quickly internalized as measured by trypsin removal from the cell surface. Once removed from the cell surface by proteolysis, regeneration of receptor activity required protein synthesis, indicating that there is no significant receptor pool within the cell. Receptor activity was also found to recycle to the cell surface after HBsAg particles were internalized.