[6,7-3H, 35S]Estrone 3-sulfate (E13S) of 3H/35S = 3.57 was incubated with female guinea pig liver slices. Small amounts of free steroid and estrone-3-glucuronide, each containing 3H, were found. In addition, E13S, 17β-estradiol 3-sulfate, and a 'disulfate' fraction, with 3H/35S = 4.4, 4.3, and 4.7, respectively, were also isolated from the incubated tissue. The latter fraction was a major metabolite and about 45% of it consisted of 'disulfates' of 16α-hydroxyestrone and estriol, thus providing strong evidence for 16α-hydroxylation in guinea pig liver slices.