The mammalian U3 snRNP is one member of a recently described family of nucleolar snRNPs which also includes U8, U13, U14, X and Y. All of these snRNPs are immunoprecipitable by anti-fibrillarin autoantibodies, suggesting the existence of a common binding site for the 34 kDa fibrillarin (Fb) protein. Two short nucleotide sequences, called Boxes C and D, present in each of these RNAs are the most likely sites for fibrillarin binding. We have developed a HeLa in vitro assembly system for binding of fibrillarin to human U3 snRNA. Reconstitution of the input RNA is specific in our assay since four of the other nucleolar small RNAs (U8, U13, X and Y) which have Boxes C and D become immunoprecipitable by anti-fibrillarin whereas two RNAs which lack these sequences (5S and 5.8S) do not. Deletion analyses of the U3 snRNA demonstrate that the presence of Box C but not Box D is required for fibrillarin binding. Moreover, seven single or double site-specific mutations in the U3 Box C abolish binding. The role of the Box C-fibrillarin interaction in the biogenesis of the Fb snRNPs is discussed.