DDX1 is a human protein which belongs to the DEAD-box protein family of enzymes and is involved in various stages of RNA metabolism from transcription to decay. Many members of the DEAD-box family of enzymes use the energy of ATP binding and hydrolysis to perform their cellular functions. On the other hand, a few members of the DEAD-box family of enzymes bind and/or hydrolyze other nucleotides in addition to ATP. Furthermore, the ATPase activity of DEAD-box family members is stimulated differently by nucleic acids of various structures. The identity of the nucleotides that the DDX1 hydrolyzes and the structure of the nucleic acids upon which it acts in the cell remain largely unknown. Identifying the DDX1 protein's in vitro substrates is important for deciphering the molecular roles of DDX1 in cells. Here we identify the nucleic acid sequences and structures supporting the nucleotide hydrolysis activity of DDX1 and its nucleotide specificity. Our data demonstrate that the DDX1 protein hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by multiple molecules: single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA molecule, a hybrid of a double-stranded DNA-RNA molecule, and a single-stranded DNA molecule. Under our experimental conditions, the single-stranded DNA molecule stimulates the ATPase activity of DDX1 at a significantly reduced extent when compared to the other investigated RNA constructs or the hybrid double-stranded DNA/RNA molecule.