The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin‐layer polyacrylamide gel isoelectric focusing in a pH 4.5–5.4 carrier ampholyte generated pH gradient, additionally containing N‐(2‐acetamido)‐2‐aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25‐hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points. Copyright © 1990 VCH Verlagsgesellschaft mbH