Using ICR strain mice as experimental animals, 0.25% (w/v) ferric ammonium citrate (FAC) was a suitable substitute for 5% (w/v) mucin as a virulence-enhancing (VE) agent in the toxicity test of Vibrio cholerae and the potency assay of cholera vaccine. There was no significant lethal toxicity difference between 0.25% FAC and 5% mucin as VE agents on Vibrio cholerae in mice. By using 0.25% FAC as a VE agent in estimating the potency of cholera vaccine, the relative potency (R.P.) of the tested cholera vaccine (Inaba) to the reference cholera vaccine (Inaba) was 0.896 +/- 0.208, the variation coefficient (V.C.) was 0.232, the correlation between the immune dose and the ratio of mouse death (r2) was 0.9932; for the cholera vaccine (Ogawa), R.P. was 1.373 +/- 0.366, V.C. was 0.266 and r2 was 0.8231. There was no significant difference between 5% mucin and 0.25% FAC used as VE agents in potency assay of cholera vaccine.