One hundred eleven laboratories, using 21 different methods based on five different principles, determined glycohemoglobin (GHb) percentages in two identical series of six lyophilized hemolysates and three similarly processed calibrators, distributed 3 months apart. To assign GHb percentages to calibrators, we used HbA1c results from nine participants who used the Bio-Rad Diamat high-performance liquid chromatographic method. Three-point calibration with assigned values improved mean intralaboratory variation (CV) from 6.6% to 3.5%. For samples with low (5.5%) and high (14.1%) GHb percentages, respectively, calibration decreased interlaboratory variation per method (from 10% to 4% and from 6% to 3%), inter-method variation (from 18% to 4% and from 16% to 3%), and overall interlaboratory variation (from 25% to 7% and from 15% to 4%). Without calibration, 71% of the laboratories did not meet the clinically desirable intralaboratory CV of 3.5%; calibration reduced this proportion to 39%. We conclude that, irrespective of the analytical method used, calibration greatly reduces all sources of GHb variation.