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Using a simian virus 40/adenovirus 12 (Ad12)-recombinant virus, the Ad12 early region 1B (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis, we have shown that the Ad12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Ad12-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immunohistochemical studies suggest that, at early times post-infection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.