The identification and partial purification of adenovirus type 5 (Ad5)-induced early proteins were achieved. Mock- and virus (high multiplicity)-infected HeLa cells were labeled for 90 min with [35S]- or [3H]methionine at 5 hr after infection in the presence of cytosine arabinoside. Labeled polypeptides from these cells were sequentially fractionated with a four-step extraction procedure: (1) 10 mM NaCl, (2) 1.7 M NaCl, (3) 2% Triton X-100, and (4) 6 M urea plus 2% Triton X-100. At each step, polypeptides from mock-infected and infected cell fractions were analyzed by DEAE-Sephadex chromatography and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-disc-PAGE) followed by autoradiography. Six virus-induced early proteins were discerned: El, 42K (E1A), E2, E2A, E2B, and E3. Polypeptides of Ad5 virions and top components and polypeptides induced at the late stage of adenovirus type 5 infection were analyzed by SDS-PAGE. They were similar to the polypeptide constituents of adenovirus type 2 (Ad2), except that the hexon and fiber of type 5 had mobilities in polyacrylamide (PA) different from those of type 2. The results eliminate the possibility that polypeptides taking part in Ad5 morphogenesis differ greatly from those of Ad2.