In order to evaluate the suprahypothalamic neuroanatomical sites of relevance to adrenocorticotropic hormone (ACTH) and cortisol regulation, single photon emission computed tomography (SPECT) investigation of induced sadness was combined with a linear regression analysis of these hormone levels during mood induction. Images from eight healthy women were analyzed by statistical parametric mapping (SPM), replicating many findings from prior sadness induction studies. Statistical parametric mapping endocrine covariate analysis showed that the ventromedial pre-frontal, anterior cingulate, and insular cortices may regulate ACTH and the insular cortex may be related to regulation of cortisol during sadness. Dysfunction of these sites may contribute to the cortisol dysregulation observed in some subjects with major depression.