Abstract Fragments of the DNA of bacteriophage ∅X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The ∅X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of ∅X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the ∅X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the ∅X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (−) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.