RNA helicase II/Gu (RH-II/Gu) is a nucleolar DEAD-box protein that unwinds double-stranded RNA and introduces secondary structure to a single-stranded RNA. We recently identified its paralogue, RH-II/Guβ, in contrast to the original RH-II/Guα. Their similar intron–exon structures on chromosome 10 suggest gene duplication. To determine functional differences, their expression, localization, and enzymatic activities were compared. RH-II/Guα is expressed two- to threefold more than RH-II/Guβ in most tissues. Both proteins localize to nucleoli, suggesting roles in ribosomal RNA production, but RH-II/Guβ also localizes to nuclear speckles containing splicing factor SC35, suggesting possible involvement in pre-mRNA splicing. The C-terminus responsible for nuclear speckle localization of RH-II/Guβ contains an arginine–serine-rich domain present in some RNA splicing proteins. In vitro assays show weaker ATPase and RNA helicase activities of RH-II/Guβ. RH-II/Guα unwinds RNA substrate with a 21- or 34-nt duplex and 5′ overhangs, but RH-II/Guβ unwinds only the shorter duplex. Although RH-II/Guβ has no RNA folding activity, it catalyzes formation of an RNA complex with unidentified structure, which is not observed when assayed with a mixture of the two enzymes. Instead, the presence of RH-II/Guβ stimulates RH-II/Guα unwinding activity. Our data suggest distinct and complex regulation of expression of the two paralogues with nonredundant gene products.