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By combining anion-exchange membrane purification with high-performance size-exclusion chromatography (HPSEC) analysis, a two-step chromatographic method was developed for the determination of vitellogenin (Vtg) in fish plasma. Most plasma protein interferences can be removed during anion-exchange membrane purification process. Vtg is eluted from the size-exclusion chromatography column with a retention time of about 9 min and is characterized based on the native molecular weight, with a limit of quantification of 20 μg Vtg mL-1 plasma. The spiked recovery and interassay variability were better than 80% and 4.8%. This method was successfully applied to analyze the plasma Vtg levels of loach (Misgurnus angaillicaudatus) and sea catfish (Enchelyopus elongatus). In addition to all the female fish, Vtg is detected in 75% of male loaches and 100% of male sea catfish. The result indicates that some chemicals or unknown factors with estrogenic activity have induced male fish to produce Vtg. [ABSTRACT FROM AUTHOR]