Egg chorion precursors (zona radiata proteins; Zrps) were purified from the blood plasma of female Atlantic cod (Gadus morhua) by salting-out and column chromatography. The salting-out procedure employed a relatively low (30%) concentration of saturated ammonium sulfate. This was a critical step that separated Zrps from approximately 89% of other plasma proteins. Subsequently, three subtypes of Zrp (Zrp-α, -β and -γ) were purified by four (Zrps-α, -γ) or five (Zrp-β) serial column chromatography steps. The Intact masses of purified Zrp-α, -β and -γ were 290 kDa, 134 kDa, and 73 kDa, while masses estimated by SDS-PAGE were 78 kDa, 54 kDa, and 47 kDa, respectively. Antibodies were prepared against Zrp-β and -γ and utilized to develop specific immunoassays. The plasma levels of Zrp-β and -γ In reproductive female cod were estimated to be 591.42±77.59 µg/ml and 768.71±120.39 µg/ml, respectively. Thus, practical procedures for the separation of Zrp subtypes were developed in cod, which resulted in the development of subtype-specific Zrp immunoassays in this species; a similar method could be adopted for the separation, detection, and quantification of Zrp subtypes in other teleosts.