An isocratic high-performance liquid chromatographic method was developed, optimized and validated for the determination of nystatin in human saliva (UV and fluorescence detection). A reversed-phase Luna C18 column (25 degrees C), with a mobile phase of MeOH, H2O, and DMF (70:20:10, v/v/v), and a flow-rate of 0.8 ml/min were used. The elution time for nystatin was 5.8+/-0.2 min. Calibration curves in human saliva were linear from 0.78 to 50 microg/ml. Limits of quantification were 0.78 microg/ml and 0.75 microg/ml for UV and fluorescence detection, respectively. The accuracy and precision values of intra- and inter-day variation studies were within acceptable limits, according to FDA guidelines. The described method has proved to be useful to give accurate measurements of nystatin in real samples.