We recently cloned a full-length DNA copy of the dengue type 4 virus genome. Analysis of the 5' terminal nucleotide sequence suggested that the three-virion structural proteins are synthesized by proteolytic cleavage of a polyprotein precursor which is encoded in one open reading frame. We now present the remaining sequence of the dengue type 4 virus genome which codes for the nonstructural proteins. The entire genome, which is 10,644 nucleotides in length, contains one long open reading frame which codes for a single large polyprotein 3386 amino acids in length. Alignment of the dengue nonstructural protein sequence with that of other flaviviruses, including yellow fever and West Nile viruses, revealed that significant homology exists throughout the entire nonstructural region of the dengue genome and this allowed tentative assignment of individual nonstructural proteins in the following order: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5-COOH. Processing of the nonstructural proteins appears to involve two types of proteolytic cleavage: the first occurs after a long hydrophobic signal sequence and the second occurs at a junction between two basic amino acids and a small polar amino acid. A notable exception is the cleavage at the N-terminus of the dengue NS3 which may take place at the junction between Gln-Arg and Ser. Comparative analysis suggests that dengue NS3 and NS5 may be involved in enzymatic activities related to viral replication and/or transcription. Putative nonstructural proteins NS2a, NS2b, NS4a, and NS4b are extremely hydrophobic, suggesting that these proteins are most likely associated with cellular membranes.