Tetracycline has been determined in human serum samples by a combination of: (1) synchronous fluorescence spectra of whole sera treated with Mg2+, and (2) the multivariate calibration methods of partial least-squares (PLS-1) and a variant of the recently introduced hybrid linear analysis (HLA), which does not require the knowledge of pure-component spectra. The calibration set was designed with 50 sera spiked with concentrations of tetracycline in the range 0.0-4.0 micrograms mL-1'. Studies concerning validation, precision, accuracy and figures of merit (selectivity, sensitivity and limit of determination) were also carried out. A novel wavelength-selection procedure was applied to minimize the effect of nonmodeled interferents present in serum samples containing bilirubin, triglycerides, and salicylate. Overall, the performance of the newly developed HLA approach seems to be better than that of PLS-1.