The human U1A protein-U1A pre-mRNA complex and the relationship between its structure and function in inhibition of polyadenylation in vitro were investigated. Two molecules of U1A protein were shown to bind to a conserved region in the 3' untranslated region of U1A pre-mRNA. The secondary structure of this region was determined by a combination of theoretical prediction, phylogenetic sequence alignment, enzymatic structure probing and molecular genetics. The U1A binding sites form (part of) a complex secondary structure which is significantly different from the binding site of U1A protein on U1 snRNA. Studies with mutant pre-mRNAs showed that the integrity of much of this structure is required for both high affinity binding to U1A protein and specific inhibition of polyadenylation in vitro. In particular, binding of a single molecule of U1A protein to U1A pre-mRNA is not sufficient to produce efficient inhibition of polyadenylation.