The effect of phenobarbital (PB) on serum hormones in late pregnancy was investigated. 18 pregnant women were studied before and after 2 weeks of PB treatment (100 mg per os, daily at 8 p.m.) by measuring serum estradiol, estriol, progesterone, testosterone, prolactin and cortisol concentrations. 7 pregnant women without PB therapy served as controls. Estradiol levels in the morning decreased, whereas those of estriol increased both in the morning and evening during PB treatment. The estriol/estradiol ratio increased in women on PB. PB affected the diurnal variation of hormones; the decrease in estradiol concentration between 8 a.m. and 8 p.m. was eliminated and the decrease in estriol concentration became more clear. PB did not significantly affect the serum levels of the other hormones investigated. As an enzyme-inducer PB may affect the fate of sex hormones.