Soybean nodules contain four major leghemoglobin (Lb) components, Lba, Lbc1, Lbc2 and Lbc3. A sensitive and selective method for quantitative analysis of the four Lb components was devised with capillary isoelectric focusing (CIEF). The changes in the concentrations of four Lb components in nodules during the initial stages of development were compared between hypernodulating soybean mutant NOD1-3 and its parent cv. Williams. The hydroponically cultivated soybean plants were periodically sampled. All the visible nodules were collected from the roots, and then the four Lb components in the largest nodules were analyzed with the CIEF method. In NOD1-3 Lbs were initially detected at 19 days after sowing (DAS), a few days earlier than in Williams at 22 DAS. The Lbcs (Lbc1, Lbc2 and Lbc3) were the main component at the earliest nodule growth stage, and the relative proportion of Lba increased with nodule growth in both NOD1-3 and Williams. This result is in agreement with previous observation, and the CIEF method is considered to be useful for Lb components analysis to define their function and gene expression.