A lymphoid T-cell line (H1591) was established by infecting peripheral blood mononuclear cells from a cotton top marmoset with Herpesvirus saimiri OMI. Analysis of these in vitro-immortalized cells revealed nonintegrated, covalently closed circular viral DNA molecules in high multiplicities with substantial rearrangements and large deletions in their L-DNA (unique) regions. One subline, designated H1591 Er, contained circular viral DNA with one stretch of H-DNA (repetitive) and one of L-DNA; the L-DNA segment consisted of a linear fusion of a 53.2-kilobase-pair piece of L-DNA (left half of L-DNA) with a 15.2-kilobase-pair L-DNA fragment from the right end of the L-DNA region. The other subline, H1591 S, contained two short regions of L-DNA, each derived from the extreme ends of virion L-DNA. Both L-DNA regions of H1591 S cells contained inverted repetitions (15.0 +/- 0.2 and 9.1 +/- 4.7 kilobase pairs). The extensive deletions of L-DNA sequences in cell line H1591 indicate that at least 73% of the genetic information in H. saimiri is not required to maintain the persistence of viral DNA and the state of transformation in lymphoid T-cells.