The enteric adenovirus type 40 (strain Dugan) grows well in tissue culture only when the E1B 55K protein of Ad5 or Ad12 is supplied in trans, either constitutively expressed in an established cell line or by coinfection with an appropriate helper virus (V. Mautner, N. Mackay, and V. Steinthorsdottir, 1989, Virology 171, 619-622). The synthesis of Ad40 E1B mRNAs and proteins has been examined under permissive and nonpermissive conditions: At late times postinfection in permissive cells, E1B-specific mRNA species of 22 and 13-14 S are made, as well as 15 and 9 S messages for the late IVa2 and ppIX proteins. None of these are detected before the onset of DNA replication and none of them accumulate in the presence of a cytosine arabinoside block to DNA replication. The failure to detect cytoplasmic mRNAs as early times cannot be attributed to a failure of mRNA transport from the nucleus as there is no accumulation of nuclear E1 RNA. In nonpermissive Hela cells only traces of E1B- and ppIX-specific mRNAs are detectable, at very late times postinfection. Antibodies raised to synthetic oligopeptides corresponding to the N- and C-terminal domains of the putative E1B 19K and 55K proteins show a high titer against the cognate peptide by ELISA, but only the E1B 19K C-terminus-specific sera have detected a unique polypeptide in Ad40-infected cells, at late times postinfection. There is no shut-off of host protein synthesis in permissive cells, despite the expression of Ad2 55K protein.