Banzi is a mosquito borne flavivirus which belongs to the Uganda S serocomplex. No nucleotide sequence data have previously been reported from any virus of this serocomplex. We have determined the nucleotide sequence of the NS5 gene from Banzi virus and the predicted amino acid sequence was elucidated. Previously identified conserved RNA polymerase, methyltransferase and flavivirus NS5 amino acid motifs were present in the Banzi virus NS5 protein. These data add to the evidence for the functional importance of the regions. The encoded amino acid sequence was compared with the predicted amino acid sequence of other flavivirus NS5 proteins. Analysis of these sequences suggested that Banzi virus is most closely related to the mosquito-borne flaviviruses and, in particular, yellow fever virus. This pattern of similarity is in accordance with the previously suggested serological classification of flaviviruses.