We have compared the deduced envelope (E) protein sequences of two biologically well-characterized yellow fever (YF) virus vaccine strains. The 17DD strain has been produced in Brazil for more than 50 years and used to successfully vaccinate millions of people worldwide. The 17D-213 is a candidate vaccine strain produced in tissue culture which has previously passed the monkey neurovirulence assay for testing human YF vaccines. Nucleotide sequence analysis of polymerase chain reaction-amplified cDNA revealed a number of mutations which were strain- and substrain-specific. A major difference of 17DD and 17D-213 as compared to 17D-204 and Asibi was the existence of a potential N-linked glycosylation site located at amino acid residues 153 and 151 of 17DD and 17D-213, respectively. These acceptor sites are apparently utilized for the addition of high-mannose carbohydrate chains as shown by endoglycosidase analyses of immunoprecipitated E proteins. Glycosylated E protein is also used to assemble YF vaccine virions. This work and eventual complete nucleotide sequence analysis of both vaccine strains should help to define possible changes involved in YF virus attenuation and allow their biological importance to be determined using a recently developed system for generating YF virus from cDNA. In addition, these data provide an estimate on the extent of genetic variability among YF 17D seeds and vaccines.