Ab responses directed against several ribonucleoprotein (RNP) Ags are a characteristic feature of systemic lupus erythematosus (SLE). Previous work in our laboratory using mouse model systems had revealed that both epitope spreading and inherent cross-reactivity between ribonucleoproteins contributes to the observed multiple specificities in autoimmune sera. We have now extended these studies to human autoimmune responses. Using purified polyclonal and mAbs derived from SLE patients, cross-reactivity between Ro60 and SmD was demonstrated. The cross-reactive epitope was mapped to nonhomologous regions on Ro60(481-505) and SmD(88-102). Five mAbs specifically recognized apoptotic cells, demonstrated variable levels of cross-reactivity toward other nonhomologous ribonucleoprotein targets and bound multiple, nonoverlapping and nonhomologous epitopes on Ro60. Our study demonstrates that cross-reactivity between frequently targeted autoantigens is an important aspect of human systemic autoimmune responses. The presence of multiple cross-reactive epitopes on Ro60 might be important for the generation of anti-Ro60 Ab in SLE patients and in normal individuals displaying no evidence of clinical disease.