Systemic lupus erythematosus (SLE) frequently involves the pleura with resultant pleural effusion. Previous studies have reported that detection of antinuclear antibodies (ANA) in pleural fluid using animal tissue as substrate was a sensitive and specific method for distinguishing SLE pleural effusions from other etiologies. The HEp-2 ANA, which uses a human cell line as substrate, is now the preferred ANA test; however, to our knowledge, no studies on pleural fluid using this assay have been reported. To determine its sensitivity and specificity, when measured in pleural fluid, HEp-2 ANA levels were determined in pleural effusion samples associated with a variety of different etiologies, including SLE, malignancy, congestive heart failure, pneumonia, tuberculosis, and a miscellaneous group of diseases. Pleural fluid ANA results were positive in 14 of 82 samples. Six of the eight (75 percent) pleural fluid samples collected from patients with SLE were ANA positive, and all but one had high titers (> 1:160) with a homogenous staining pattern. The remaining two patients with SLE with negative pleural fluid ANA had recurrent pulmonary emboli and congestive heart failure, rather than lupus pleuritis. Eight of 74 patients (10.8 percent) without clinical evidence of SLE had a positive pleural fluid ANA, with the majority having a speckled pattern. High titers were noted in three. These results indicate that a negative or low titer ANA and a speckled staining pattern in pleural fluid from a patient suspected of lupus pleuritis suggest an alternative diagnosis. High pleural fluid titers (up to 1:640) were seen occasionally in patients with inflammatory pleural effusions in the absence of SLE.