Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoencephalopathy vanishing white matter with a wide clinical spectrum. eIF2B comprises five subunits (α–ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide‐exchange factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional effects of selected VWM mutations in EIF2B2–5 by coexpressing mutated and wild‐type subunits in human cells. The observed functional effects are very diverse, including defects in eIF2B complex integrity; binding to the regulatory α‐subunit; substrate binding; and GEF activity. Activity data for recombinant eIF2B complexes agree closely with those for patient‐derived cells with the same mutations. Some mutations do not affect these parameters even though they cause severe disease. These findings are important for three reasons; they demonstrate that measuring eIF2B activity in patients' cells has limited value as a diagnostic test; they imply that severe disease can result from alterations in eIF2B function other than defects in complex integrity, substrate binding or GEF activity, and last, the diversity of functional effects of VWM mutations implies that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B, not restoring eIF2B activity. Hum Mutat 32:1036–1045, 2011. © 2011 Wiley‐Liss, Inc.