A reagentless amperometric biosensor sensitive to lactate was developed. This sensor comprises a carbon paste electrode modified with lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD(+)) cofactor and Meldola's blue (MB) adsorbed on silica gel coated with niobium oxide. The amperometric response was based on the electrocatalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of lactate with NAD(+) under catalysis of LDH. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, LDH and NAD(+) amounts and applied potential. The biosensor showed an excellent operational stability (95% of the activity was maintained after 250 determinations) and storage stability (allowing measurements for over than 2.5 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing lactate quantification at levels down to 6.5 X 10(-6) mol L-1. Moreover, the biosensor showed a wide linear response range (from 0.1 to 14 mmol L-1 for lactate). These favorable characteristics allowed its application for direct measurements of lactate in biological samples such as blood. The precision of the data obtained by the proposed biosensor show reliable results for real complex matrices.181212081214