Although estimated average requirements for biotin have been proposed, the human requirements for biotin in specific populations and at various ages remain uncertain, in part because indicators of biotin status have not been validated. With the use of improved methods for measuring biotin and metabolites, a recent study indicated that decreased urinary excretion of biotin and bisnorbiotin is an early and sensitive indicator of biotin deficiency, but decreased serum concentration of biotin is not. Increased urinary excretion of 3-hydroxyisovaleric acid (3-HIA), a leucine metabolite that is excreted in increased quantities with deficiency of the biotin-dependent enzyme beta-methylcrotonyl-CoA carboxylase, is also an early and sensitive indicator of biotin deficiency. When these indicators were assessed longitudinally in 13 pregnant women, biotin excretion was not significantly decreased early in pregnancy but did decrease significantly from early to late pregnancy. Excretion of 3-HIA was abnormally increased in about three-fourths of the women studied in both early and late pregnancy. Thus, each indicator detected biotin deficiency late in pregnancy, but assessment of biotin status for the two indicators conflicted early in pregnancy. Preliminary results from a trial assessing response of 3-HIA excretion to biotin treatment indicate that biotin status is indeed impaired both early and late in pregnancy.