In this laboratory, Raghupathy et al. (1963, 1964) demonstrated the incorporation of 14C-amino acids into protein by (a) slices prepared from the thyroid glands of rats, guinea pigs and sheep and (b) monolayer cultures of isolated thyroid gland cells. In vitro protein synthesis by subcellular fractions of the thyroid gland, however, has not hitherto been studied. Since the early report of McLean et al. (1958), considerable evidence has accumulated demonstrating the incorporation of amino acids into protein by mitochondria isolated from a variety of animal and plant tissues as well as from microorganisms ( Simpson, 1962). The present communication deals with the incorporation of 14C-amino acids into protein by mitochondria isolated from sheep thyroid glands. An attempt has been made to elucidate the steps involved in this process by studying the cofactor requirements for and the effects of various inhibitors, such as DNase, ∗ ∗ Abbreviations: DNase for deoxyribonuclease; RNase for ribonuclease; ATP for adenosine triphosphate; GTP for guanosine triphosphate; PEP for phosphoenol pyruvic acid; PK for pyruvate kinase; RNA for ribonucleic acid; DNA for deoxyribonucleic acid; FDNB for 1-fluoro-2,4-dinitrobenzene; EDTA for ethylenediamine tetraacetic acid; GSH for glutathione; Tris for tris (hydroxymethyl) aminomethane; U for uniformly labeled; DNP for dinitrophenyl. RNase, actinomycin D and puromycin, on the amino acid incorporating activity of thyroid mitochondria.