Strains of C. trachomatis representative of 14 serotypes were grown in HeLa 229 [human cervical carcinoma] cells. HeLa cell susceptibility to chlamydial infection was increased by treating the host cells with DEAE-dextran. Optimal conditions of DEAE-dextran treatment were determined for each serotype of C. trachomatis to maximize chlamydial yields. Chlamydial polypeptides were selectively radiolabeled with 3H-labeled amino acids in the presence of emetine, an inhibitor of HeLa cell protein synthesis. The radiolabeled chlamydiae were purified from host cell components by density gradient centrifugation and their polypeptide composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of the major chlamydial polypeptide of 38,000-42,000 daltons among the different serotypes correlated closely with the predominant human infections caused by each serotype. Lymphogranuloma venereum agents possessed a unique polypeptide of 118,000 daltons not found among trachoma inclusion conjunctivitis strains of chlamydiae. Chlamydial surface polypeptides were selectively radiolabeled with 125I by lactoperoxidase-catalyzed oxidation. The major chlamydial polypeptide and polypeptides of 155,000 and 29,000 daltons were thus identified as surface polypeptides of the chlamydial elementary body. Apparently, the 155,000 dalton polypeptide is a species-specific antigen, the major polypeptide is the principal outer membrane protein, and the 29,000 dalton polypeptide is the type-specific antigen.