A nuclear membranous fraction prepared from a DNase digest of calf thymus nuclei was purified by continuous sucrose-gradient centrifugation. Two nuclear membrane fractions were found to localize at buoyant density 1.19 and 1.21, and to sediment significantly deeper than mitochondria treated with DNase. Structurally, the purified nuclear membranes were sheets of membrane and large vesicles having a partial bilaminar structure. The chemical composition of the nuclear membranes was determined and compared with those of mitochondria and microsomes prepared in parallel. Low-temperature spectra of the nuclear membranes showed that the nuclear membrane cytochrome system of calf thymocyte is composed at least of a + a 3, c, c 1, and b types including b 5 and b 557–558.5.