Hypothalamic tyrosine hydroxylase (TH) activity of ovariectomized rats is modulated by gonadal steroids in vivo. The present study investigated the effects of ovariectomy and subsequent replacement of progestational and nonprogestational steroids on TH activity in vitro. All progestational steroids tested inhibited TH activity. Saturation of the A-ring of the steroid nucleus reduced apparent inhibitory activity. Kinetic studies revealed that both Vmax and Km were virtually unaltered for the substrate tyrosine in the presence of progesterone. Progesterone, in a dose dependent manner, increased the Km for the cofactor, DMPH4. These results suggest that progestational steroids decrease apparent TH activity by inhibiting the enzyme-reduced pteridine cofactor complex. (Endocrinology93: 1453, 1973)