Demeclocycline (DM) and methacycline (MT) have been determined by europium-sensitized fluorescence, using EDTA as co-ligand and cetyltrimethylammonium chloride as surfactant. The methods have been developed in slightly alkaline solutions, with the formation of a new chelate where the lanthanide ion is bound to the beta-diketone group. Calibration graphs between 0.01 and 0.1 microg mL(-1) have been obtained for DM and MT determination. Both methods have been applied to the determination of these tetracyclines in serum samples with satisfactory recovery results.