NAP57 has been found as a component of nuclear matrix protein complex with ability to specifically bind alphoid DNA. Polyclonal antibodies against NAP57 were raised in order to investigate intranuclear localization and interactions of the protein. Two types of localization were observed: a) nucleoplasmic and b) nucleolar. A bulk of nucleoplasmic fraction is present in splicing factors compartments (SFC). The type of localization pattern does not depend on the cell cycle phase, but we revealed changes in NAP57 localization pattern during S phase. According to immunoprecipitation and immunofluorescence assays, NAP57 specifically interacts with DEAD RNA helicase p68 in vitro and co-localizes with helicase p68 in the nucleus of cultured cells. We suppose participation of both proteins in processing of small nuclear RNA on the SFC periphery, and positioning of the nucleolus according to centromere regions of chromosomes.