Analysis of cellular extracts of HeLa cells infected with adenovirus type 2 (Ad2) by immunoprecipitation with antiserum against the late nonstructural 100,000-dalton (100K) protein revealed the presence of a specific complex between the 100K protein and newly synthesized hexon molecules. Serological analysis of the hexon molecule in the 100K/hexon complex with antibodies specific for hexon monomers or trimers showed that only monomeric hexon molecules were associated with the 100K protein. By immunofluorescence microscopy this monomeric hexon was primarily found in the cytoplasm, whereas the trimeric form was mainly confined to the nucleus of infected cells. We conclude that in the cytoplasm of Ad2-infected cells newly synthesized, monomeric hexon molecules can interact with the 100K protein. This suggests that the 100K protein may play some role either in trimerization of newly synthesized, monomeric hexon molecules and/or in its transport from the cytoplasm into the nucleus.