Vitamin B ₁₂ (VitB ₁₂, cobalamin) deficiency has been associated with various neuropsychiatric conditions, such as peripheral neuropathy, subacute combined degeneration, affective disorders, and cognitive impairment. Current assays analyze vitamin B ₁₂, of which only a small percentage is metabolically active. Measurement of its active fraction, holotranscobalamin, might be of greater relevance, but data in populations with neuropsychiatric populations are lacking. In this study, in order to validate VitB ₁₂ and holotranscobalamin (holoTC) serum levels for the detection of VitB ₁₂ deficiency in neuropsychiatric conditions, we compared the validity of VitB ₁₂ and holoTC in a patient cohort with neuropsychiatric conditions suspicious for VitB ₁₂ deficiency. The cohort included all patients admitted to the Department of Neurology at our university between 2005 and 2009 with at least two parameters of the VitB ₁₂ metabolism available (n = 1,279). We used elevated methylmalonic acid as the external validation criterion for VitB ₁₂ deficiency and restricted our analyses to subjects with normal renal function. Among all normal renal function patients, 13.2% had VitB ₁₂ deficiency. In receiver operating characteristic curve (ROC) analysis, correlation of VitB ₁₂ and holoTC with vitamin B ₁₂ deficiency was generally weak, and the areas under the curve (AUC) were not significantly different for holoTC compared to vitamin B ₁₂ in all subjects (AUC: 0.66 [95%CI: 0.51-0.82]; p = 0.04 vs. 0.72 [0.65-0.78], p < 0.0001) and in subcohorts of patients with classical VitB ₁₂ deficiency syndromes. The positive predictive values for holoTC and vitamin B ₁₂ were low (14.7 vs. 21.0%) and both were associated with more false-positive than true-positive test results. holoTC does not show superior diagnostic accuracy compared to VitB ₁₂ for the detection of VitB ₁₂ deficiency in subjects with neuropsychiatric conditions. Neither test can be recommended to diagnose VitB ₁₂ deficiency in subjects with neuropsychiatric disorders.