In experiment 2, 20 crossbred cycling gilts were randomly allotted to two treatments: riboflavin adequate (control) and riboflavin deficient cornstarch-soybean meal diets. Gilts were checked daily for estrus. Blood was obtained by venipuncture at 1000 h every 2 weeks after the establishment of the deficiency. Plasma was frozen at -20°C and assayed for progesterone (P₄) and estradiol-17 (E₂). After 28 days, the first urine sample from two consecutive days was collected and assayed for estrone sulfate (E₁ S) after enzymatic hydrolysis. Gilts on the riboflavin deficient diet became anestrous 63 days after the beginning of the experiment. The average time elapsed between consecutive estrous periods increased with length of time on the deficient diet. The control gilts had elevated (P<0.01) plasma P₄ levels between Days 4-18 with peak concentrations of 28.3 ± 1.0 ng/ml on Day 12 of the cycle. In contrast, P₄ levels in riboflavin deficient gilts were significantly elevated (P<0.001) over control gilts (64.2 ± 1.0 ng/ml). The E₂ plasma concentrations in control gilts were low (19.6 ± 1.9 pg/ml) from Days 2-18, but were elevated (64.7 ± 2.3 pg/ml; P<0.01) 2 days before the onset of estrus. The riboflavin deficient gilts displayed significantly elevated E₂ levels (82.6 ± 1.8 pg/ml; P<0.001). The E₁ S urine concentrations were elevated in the control gilts with peak levels of 22.1 ± 2.3 ng/mg creatinine on Day 20. The riboflavin deficient guts had low E₁ S levels throughout the test period (4.1 ± 0.8 ng E₁ S/mg creatinine). These data indicate a loss of ovarian cyclicity and the presence of abnormal plasma concentrations and urinary excretion of sex steroids for riboflavin deficient gilts.