AbstractGroup specific component (Gc) is known to interact with both vitamin D3metabolites and actin. The present study was undertaken to determine if analytical isoelectric focusing could form the basis of a simple and reliable method for discriminating native Gc and complexes with 25‐hydroxycholecalciferol (25(OH)D3) from those formed with actin and with both ligands. Increasing amounts of G‐actin and/or 25(OH)D3were added to purified Gc of both Gc1 and Gc2 phenotypes. Actin and 25(OH)D3interacted independently and simultaneously with native Gc, giving rise to three different complexes of increasing acidity: Gc‐25(OH)D3, Gc‐actin, and actin‐Gc‐25(OH)D3, which were clearly resolved from the native Gc protein and from each other. In addition, the inherent differences in microheterogeneity and polymorphism between the two major phenotypes examined (Gc1 and Gc2) were also retained in their respective complexes. Similar results were obtained upon addition of 25(OH)D3or actin to whole human serum, the bands corresponding to native or complexed Gc being recognized in this case by print immunofixation. These results indicate that complexes formed between Gc protein and both 25(OH)D3and actin can be clearly detected and resolved by electrophoretic