A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of α, β and γ polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 ± 2,500, 80,500 ± 2,000 and 30,000 ± 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the α chain. The presence of dual α chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine α chains is ~ 1,800 daltons. On activation of bovine C4 both α chains were cleaved into α' chains (87,000 and 85,000 daltons) characteristic of C4b.