Objectives: Evaluation of the analytical performance of a biosensor for the measurement of lactate in whole blood samples and comparison of the results of patients samples with a routinely used spectrophotometric enzymatic method.Design and Methods: Heparinized whole blood samples of patients and aqueous control samples were used to determine precision and carry-over. For comparison of the lactate biosensor and the enzymatic method human blood samples were split and measured.Results: Satisfactory within-run (n = 7) and day-to-day (n = 15) precision was found, while carry-over was minimal (<0.2%). A statistically significant relation between the lactate levels in whole blood samples (n = 31) and the corresponding plasma samples was found: y = 0.98x - 0.05 and r = 1.00. No correlation was found between the hematocrit (range 0.23-0.51) and the difference in lactate concentration between plasma and whole blood. When comparing patient results (n = 722) obtained with the spectrophotometric method and the biosensor method, the biosensor measured 13% higher lactate levels. A correction on the basis of the hematocrit minimized this difference.Conclusion: The combination of analytical performance, easy handling, rapid analysis, and measurement in whole blood makes the biosensor suitable for lactate STAT-analyses. Care must be given to the interpretation of the results as well as to the preanalytical aspects.