Peripheral plasma estrone sulfate (E(1)-S) concentrations were characterized throughout gestation in singleton and twin bearing cows by a direct radioimmunoassay method. Maternal plasma E1-S was detectable from around day 100 and after that its concentration increased progressively to term in both singleton (n = 5) and twin bearing (n = 5) cows. Twin bearing cows had a significantly higher E1-S concentrations in some time from mid-gestation to term when compared to singleton cows. E1-S concentration in the twin bearing cows increased rapidly in the third trimester and peaked (16.7 ng/ml) on the day of calving. In the singleton cows the concentration increased gradually and peaked (7.1 ng/ml) about 10 days before calving and then subsequently decreased. Our results indicate that singleton and twin bearing cows show a disparate E1-S profile from mid-gestation to term.