Cells infected with human adenovirus type 5 have been labelled with 32P-orthophosphate under various conditions and extracts examined, after denaturation in sodium dodecyl sulphate (SDS), by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. A number of polypeptides appear to be phosphorylated specifically as a result of infection. Early in infection, phosphorylation of a polypeptide of apparent mol. wt. 26 K associated with ribosomes can be detected. Two other phosphorylated polypeptides of apparent mol. wt. 72 K and 18 K can also be seen, the former being mainly confined to the nucleus and capable of being precipitated by the previously described virus-specific P antiserum. The 18 K phosphorylated polypeptide is found mainly in association with membrane fractions. Later in infection phosphorylated polypeptides of apparent mol. wt. 100 K and 39 K can be recognized, the former being associated with ribosomes but removed, however, with a high salt wash; the latter component is mainly detected in the nucleus. Analysis of the purified 32P-labelled virus by the SDS PAGE technique indicated that a structural polypeptide of apparent mol. wt. 66 K (IIIa) was also phosphorylated.