The flavivirus genome codes for structural and nonstructural proteins which are produced from a common polyprotein precursor by proteolytic processing. The nonstructural protein NS1 is hypothesized to be involved in virus assembly and maturation as well as in protective immunity. This paper describes the synthesis of several forms of yellow fever (YF) virus NS1 protein during virus cultivation in vitro. These include cell-associated and secreted NS1 forms, the major difference among these being their N-linked glycosylation status. High-mannose and complex (or hybrid)-type moieties were identified on YF NS1 by analysis with endoglycosidases of known specificity. These studies are the basis for a systematic approach to determining the role of NS1 in the virus cycle and possibly in pathogenesis.