To investigate the clinical utility of a newly developed hybrid ELISA for antinuclear antibodies (ANA), a cross-sectional study of patients admitted to the Section of Rheumatology was initiated. The ELISA was compared to indirect immunofluorescence (IIF) on HEp-2 cells. Accuracy of tests was analyzed using receiver-operating characteristic methodology (ROC). In addition, diagnostic sensitivity, specificity and predictive values were calculated for each assay. Results from the ROC analysis showed a slightly superior accuracy for IIF as compared to ELISA. Furthermore, IIF showed higher diagnostic sensitivity and positive predictive value for all combinations of patients and reference populations. This was due to enhanced detection by IIF, in contrast to ELISA, of diagnostically useful antibodies. IIF detected 87.4% and ELISA detected 84.2% of sera with antibodies against extractable nuclear antigens (ENA). In addition, IIF detected diagnostically important antibodies that are not included among the anti-ENA. The hybrid ELISA either lacks or does not contain the relevant antigens in sufficient amount. Inclusion of these antigens may further enhance the performance characteristics of the ELISA.