The site of interaction between human U1 RNA and one of its uniquely associated proteins, A, was examined with in vitro binding assays. The A protein bound directly to stem-loop II of U1 RNA in a region which exhibits sequence similarity to U2 RNA. The similarity with U2 RNA was in a region that has been shown to interact with a U2 RNA-associated protein. The A protein-binding site on U1 RNA overlapped a previously described epitope for an RNA-specific human autoantibody (S. L. Deutscher and J. D. Keene, Proc. Natl. Acad. Sci. USA 85:3299-3303, 1988), supporting the hypothesis that the anti-RNA antibody originated as an anti-idiotypic response to A protein-specific autoantibodies.