During the course of lytic infection with the adenovirus 2-SV40 hybrid Ad2+ND1 dp2, a viral mRNA is synthesized whose 5′ end includes the start of the adenovirus 2 fiber gene and whose 3′ end lies in SV40 sequences. This hybrid mRNA codes for a protein(s) of 23-24K daltons (G. Fey, J. B. Lewis, T. Grodzicker and A. Bothwell, manuscript submitted). Biochemical and electron microscope heteroduplex analyses reveal that two forms of this mRNA exist in the cytoplasm of cells at late times in infection. Both forms contain the conventional tripartite leader present on "late" adenovirus 2 mRNAs, but they differ in the presence or absence of a fourth leader sequence consisting of 180 nucleotides of adenovirus 2 sequences located 2,000 nucleotides upstream from the gene. By fractionation of total poly(A)-containing Ad2+ND1 dp2 "late" cytoplasmic RNA in methyl mercuric hydroxide-agarose gels, we have isolated both forms of the hybrid mRNA and established that the presence or absence of the fourth leader sequence has little or no influence on the ability of the messenger to direct the synthesis of the 23-24K dalton protein(s) in a cell-free translation system derived from rabbit reticulocytes. © 1978.