Two distinct nuclear antigens, designated NSpI and NSpII, have been characterized and differentiated from the centromeric antigen that reacts with sera from patients with the CREST syndrome. Both NSpI and NSpII produce a speckled pattern of indirect immunofluorescence on HEp-2 cells that resembles the pattern seen with anticentromere antibodies (ACA). They are differentiated from the ACA staining pattern by the absence of metaphase chromatin staining by NSpI antisera and by the absence of a discrete speckled pattern of staining by NSpII. Further, both NSpI and NSpII stain predominantly the peritubular nuclei of mouse kidney cryostat sections. NSpII is sensitive to trypsin, proteinase K, and HCI extraction, suggesting that it is a relatively soluble nuclear protein. NSpI was also sensitive to protease treatment but was not extracted with 0.1N HCl, suggesting that it is a tightly bound nuclear protein.