Serum from patients with scleroderma recognizes the clumpy autoantigen. The present studies addressed the issue as to whether the clumpy nucleolar autoantigen recognized by scleroderma serum is fibrillarin-U3 snoRNP. Clones encoding for clumpy autoantigen were immunodetected from a lambdagt11 HeLa cell random-primed library with the serum from a patient with diffuse scleroderma and autoautoantibodies against clumpy autoantigen. Sequences from the recombinant phages were amplified by PCR and subcloned into a pCRII vector. The DNA was sequenced by a dideoxy termination reaction. Ten lambdagt11 clumpy clones were detected by immunoscreening. One containing the glycine-rich and RNP2 fibrillarin domains was expressed in lysogenic bacteria. The recombinant proteins were used to elicit antibodies in rabbits, and these exhibited clumpy nucleolar reactivity. The recombinant fibrillarin tested by ELISA was recognized by the clumpy scleroderma serum from the majority of patients. In situ hybridization assays showed that the fibrillarin tagged by the elicited antibodies was colocalized with U3 snoRNP in the nucleolus in a clumpy manner and coprecipitated the U3 snoRNP. In conclusion, the fibrillarin-U3 snoRNP complex is the major component of the clumpy subcellular domain. Therefore these molecules constitute an important target of scleroderma autoantibodies.