Intermediates in the folate-dependent methylation pathways may play a role in the etiology and treatment of such mental disorders as major depression. These pathways include a step dependent on a riboflavin (B2)-derived coenzyme, flavin adenine dinucleotide (FAD), which is reportedly sensitive to thyroid status and to phenothiazine and tricyclic drug exposure. In a sample of 52 male and female acute psychiatric inpatients, 17% (n = 9) showed B2 deficiency (i.e., insufficient FAD activity) on a functional red blood cell enzyme assay, but only one B2-deficient individual showed deficiency in another B-complex vitamin (folate). All patients with B2 deficiency were women, who were also significantly younger than the rest of the sample. The B2-deficient women had significantly lower thyroxine levels, even when controlling for sex and covarying for age. B2-deficient patients exhibited a nonsignificant trend toward more unipolar depression (44% vs 14%), but not toward bipolar or schizophrenic disorders. As in a previous study, drug exposure did not show a relationship to riboflavin deficiency in this sample. The findings suggest that B2 (FAD) activity may serve as a sensitive marker of thyroxine status in certain female psychiatric inpatients and that B2 deficiency may play an etiological role in defects of the methylation pathways in a subset of mentally ill individuals.