We report the characterization of a human mitochondrial translation release factor, mtRF1a, based on the following criteria: (1) sequence similarity to known release factors; (2) mitochondrial localization, import, and cleavage of the preprotein; (3) presence in mitochondria of all cell types and tissues tested; (4) demonstration of release factor activity in vitro with E. coli ribosomes; (5) rescue of the Δmrf1 respiratory deficiency in fission and budding yeast in vivo; and (6) demonstration of a growth phenotype in mtRF1a-depleted human cells. The gene discussed is MTRF1L; the disease is hyperinsulinemic hypoglycemia, familial, 4.