We performed this study to determine sensitivity and specificity of pleural effusion antinuclear antibodies (ANA) at a titer of ≥1 : 160, and the ratio of pleural effusion to serum ANA of ≥1, to distinguish between pleural fluid from lupus pleuritis and other causes. A prospective study of 54 patients with pleural effusion (12 lupus pleuritis, seven parapneumonic effusion, 26 malignancy-associated pleural effusions, nine transudative effusions) was performed. ANA at a titer of ≥1 : 160 were found in 11 of 12 lupus pleuritis samples, and in four of 42 pleural effusions from non-systemic lupus erythematosus (SLE) patients. The pleural effusion ANA at a titer of ≥1 : 160 gave a sensitivity of 91.67% for lupus pleuritis, with a specificity of 83.33% when compared with all other pleural effusions, 90.91% when compared with exudative effusion (parapneumonic effusion and malignancy-associated effusion) and 55.56% when compared with the transudative pleural effusion group. Using the ratio of pleural effusion to serum ANA of ≥1, the sensitivity and the specificity decreased to 75.00% and 78.57%, respectively. This study provides further evidence that the pleural effusion ANA at a titer of ≥1 : 160 is a sensitive and specific diagnostic biomarker for lupus pleuritis in patients with lupus. However, pleural effusion ANA can occasionally be found in other conditions.