Serum and whole blood folate measurements are used to establish folate deficiency. Most methods used in clinical laboratories are automated, nonistopic methods that use folate-binding protein. Linearity, imprecision, and method comparison studies, including serum and whole blood hemolysates, were performed with the Access, Advia Centaur, ARCHITECT i2000, Elecsys 2010, and IMMULITE 2000 methods. The QuantaPhase II radioassay served as the comparison method. (Proprietary information is given in the text.) The Access and IMMULITE 2000 methods had higher systematic errors in linearity studies than the other 3 methods. The imprecision of all methods was acceptable (coefficient of variation, < 10%) even at low folate concentrations with the exception of the Elecsys 2010 (coefficient of variation, 16%). Method comparison studies using serum samples revealed calibration differences between the Access and Elecsys 2010 methods and the comparison method. Method comparison studies using whole blood samples showed poorer agreement between each of the automated methods and the comparison method than was seen with serum samples. The ARCHITECT i2000 folate assay demonstrated the best analytic performance. The poor agreement seen with whole blood hemolysates likely is due to calibration differences and differences in hemolysate preparation conditions.