The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family. It is a unique enzyme, which possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular secondary structure in single-stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity. High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity. Mg2+ is required for the helicase activity but not for the RNA-folding reaction. Affinity-purified anti-(RH-II/Gu) polyclonal Ig inhibit the RNA-unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA-folding activity of RH-II/Gu but completely inhibited the RNA-helicase activity. A mutant that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single-stranded RNA. The two enzymatic activities of RH-II/Gu reside in distinct domains. Amino acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity. Amino acids 646-801 fold single-stranded RNA but lack helicase activity. This report shows distinct RNA-unwinding and RNA-folding activities residing in separate domains within the same protein.