Ramin V. Parsey, R. Todd Ogden, Jeffrey M. Miller, Adrienne Tin, Natalie Hesselgrave, Ellen Goldstein, Arthur Mikhno, Matthew Milak, Francesca Zanderigo, Gregory M. Sullivan, Maria A. Oquendo, J. John MannBackground: Serotonin 1A receptors (5-HT1A) are implicated in major depressive disorder (MDD). We previously reported higher 5-HT1A binding potential (BPF) in antidepressant naive MDD subjects compared with control subjects, while other studies report lower BPND. Discrepancies can be related to differences in study population or methodology. We sought to replicate our findings in a novel cohort and determine whether choice of reference region and outcome measure could explain discrepancies.Methods: Nine new control subjects and 22 new not recently medicated (NRM) MDD subjects underwent positron emission tomography. BPF and BPND were determined using a metabolite and free fraction corrected arterial input function. BPND was also determined using cerebellar gray matter (CGM) and cerebellar white matter (CWM) reference regions as input functions.Results: BPF was higher in the new NRM cohort (p = .037) compared with new control subjects, comparable to the previous cohort (p = .04). Cohorts were combined to examine the reference region and outcome measure. BPF was higher in the NRM compared with control subjects (p = .0001). Neither BPND using CWM (p = .86) nor volume of distribution (VT) (p = .374) differed between groups. When CGM was used, the NRM group had lower 5-HT1A BPND compared with control subjects (p = .03); CGM VT was higher in NRM compared with control subjects (p = .007).Conclusions: Choice of reference region and outcome measure can produce different 5-HT1A findings. Higher 5-HT1A BPF in MDD was found with the method with fewest assumptions about nonspecific binding and a reference region without receptors.