A tracer dose of estradiol-17β-6,7-3H was injected intravenously into male subjects simultaneously with a14C-labeled steroid. The latter was either 16-ketoestradiol-17β-16-14C, 16-epiestriol-16-14C, or estriol-16-14C. All eight urinary metabolites of estradiol-17β-3H studied, and the main metabolites of the 16-oxygenated steroids, were predominantly split by β-glucuronidase hydrolysis. The major urinary metabolite released by solvolysis was estrone, as a conversion product of estradiol-17β-3H. After injection of estradiol-17β-3H together with either 16-ketoestradiol-17β-14C or 16-epiestriol-14C, urinary 16β-hydroxyestrone contained both isotopes. 16α-Hydroxyestrone, on the other hand, contained3H but only insignificant amounts of14C. This suggests formation of 16β-hydroxy-estrone but not 16α-hydroxyestrone from these injected 16-oxygenated steroids. 16-Epiestriol was metabolized to identifiable urinary metabolites to a considerably greater degree than was estriol. This was confirmed in a further experiment in which only 16-epiestriol-14C was injected.