The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.