Lactate dehydrogenase isoenzymes have been used to classify the nature of pleural effusion. Nevertheless, studies have reported conflicting results. The objective of this study was to evaluate the diagnostic value of lactate dehydrogenase isoenzymes in the analysis of pleural effusions. Pleural fluid samples obtained from three respective diagnostic groups: group I transudate (n = 23), group II parapneumonic effusion (n = 29) and group III malignant effusion or pleuritis carcinomatosa (n = 41) were evaluated. Total lactate dehydrogenase activity and lactate dehydrogenase (LDH) isoenzyme pattern were significantly different between transudative (group I) and exudative (group II and III) effusions. Group II and III showed a low percentage of LDH1 (p < 0.001), whereas the percentages of LDH4 (p < 0.001) and LDH5 (p < 0.001) were higher compared to group I. Moreover, in exudative effusions the percentage of LDH1 (p < 0.005), LDH4 (p < 0.005), as well as LDH5 (p < 0.005) were significantly different between parapneumonic and malignant effusions. In contrast to relative lactate dehydrogenase isoenzyme values, the absolute values of lactate dehydrogenase isoenzymes did not differ between group II and group III. Logistic regression analysis yielded a strong discrimination between group I and II+III, simultaneously using lactate dehydrogenase, glucose and protein as explanatory variables. Logistic regression analysis yielded only a weak discrimination between group II and III, simultaneously using lactate dehydrogenase, glucose and the absolute values of LDH2 and LDH4 as explanatory variables. In conclusion, the lactate dehydrogenase isoenzyme pattern differed between pleural effusions of transudative and exudative origin. However, including lactate dehydrogenase isoenzyme activities in the biochemical work-up of pleural effusions did not reveal an additional discriminatory value in the assessment of the classification of these effusions.