The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3 end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5 end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5 end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.The retroviral life cycle is characterized by the conversion of the single-stranded RNA genome found in virions into a double-stranded linear DNA that is subsequently integrated into the host cell genome. Viral DNA synthesis takes place in the cytoplasm of the infected cell. The RNA genome of the virus is copied into DNA by a virally encoded enzyme, reverse transcriptase (RT). RT contains two enzymatic activities, a DNA polymerase that can copy either an RNA or a DNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA hybrid. Like many other DNA polymerases, RT requires both a template and a primer. First (minus)-strand DNA synthesis is initiated from a cellular tRNA primer that is base paired at the primer binding site (PBS), which is near the 5Ј end of the viral genomic RNA. During the reverse transcription process, this tRNA primer is removed from the end of the minus-strand DNA by RNase H; this defines the right (U5) end of the linear viral DNA. Second (plus)-strand DNA synthesis is initiated from a polypurine tract (PPT) primer adjacent to U3. The RNase H cleavages that generate and remove PPT primer define the left (