Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884-1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243-337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 degrees C, as well as after 5 weekly freeze-and-thaw cycles at -20 degrees C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.