The influenza virus NS1 protein is the only known example of a protein that inhibits the nuclear export of mRNA. To identify the functional domains of this protein, we introduced 18 2- or 3-amino-acid substitutions at approximately equally spaced locations along the entire length of the protein. Two functional domains were identified. The domain near the amino end (amino acids 19 through 38) was shown to be the RNA-binding domain, by using a gel shift assay with purified NS1 protein and spliced viral NS2 mRNA as the RNA target. The second domain, which is in the carboxy half of the molecule, was presumed to be the effector domain that interacts with host nuclear proteins to carry out the nuclear RNA export function, by analogy with the effector domain of the Rev proteins of human immunodeficiency virus (HIV) and other lentiviruses which facilitate rather than inhibit nuclear RNA export. The NS1 protein has a 10-amino-acid sequence that is similar to the consensus sequence in the effector domains of lentivirus Rev proteins, specifically including two crucial leucines at positions 7 and 9 of this sequence. However, the effector domains of the NS1 and Rev (HIV type 1 [HIV-1]) proteins differed in several significant ways including the following: (i) unlike the HIV-1 Rev protein, NS1 effector domain mutants were negative recessive rather than negative dominant, (ii) the NS1 effector domain is about three times larger than the effector domain of the HIV-1 Rev protein, and (iii) unlike the HIV-1 protein, NS1 effector domain mutants exhibited a surprising property, a changed intracellular/intranuclear distribution, compared with the wild-type protein. These differences strongly suggest that the effector domains of the NS1 and Rev proteins interact with different nuclear protein targets, which likely explains the opposite effects of these two proteins on nuclear mRNA export.