Measurement of lactate levels is important in the care of critically ill adult and pediatric patients. We compared 3 whole blood lactate methods (Radiometer ABL 725, Radiometer Medical A/S, Bronshoj, Denmark; i-STAT, i-STAT, East Windsor, NJ; and Nova Lactate Plus, Nova Biomedical, Waltham, MA) with 2 plasma-based methods (Roche Integra, Roche Diagnostics, Indianapolis, IN; and Vitros, Ortho Clinical Diagnostics, Rochester, NY). The Vitros LAC slide assay was used as the reference method. Results were compared by least squares regression and Bland-Altmann plots and by comparing concordance within clinically relevant lactate ranges. Correlation between lactate methods was good with slopes between 0.87 and 1.06 and intercepts of 0.9 to 1.8 mg/dL (0.1–0.2 mmol/L) of lactate for all 4 methods compared with the Vitros. At high (>54.1 mg/dL [6 mmol/L]) lactate values, the Radiometer and i-STAT methods reported lower lactate results compared with the Vitros and Integra. The Nova analyzer reported higher lactate results than either the Vitros or Integra. The negative bias in i-STAT and Radiometer results may confound the interpretation of patient condition if multiple methods are used within the same institution.