The low human immunodeficiency virus type-1 (HIV-1) expressing T-cell line, ACH-2, was used to investigate accumulation of the circular, extrachromosomal form of HIV DNA (HD) after tumor necrosis factor-alpha (TNF-alpha) induction. We chose the 2 long terminal repeat (LTR) circular form to analyze unintegrated HD by polymerase chain reaction (PCR), using primer pairs which flank the 2 LTR HD. Approximately a 10-fold increase in 2 LTR HD was detected intracellularly in the TNF-alpha-induced ACH-2 cells using an end point-dilution assay. To examine the cellular compartment location of the 2 LTR HD accumulation, ACH-2 cells were fractionated into cytoplasmic and nuclear components and further subjected to PCR. A 4- to 5-fold increase in the 2 LTR HD signal was observed in the nuclear fraction. These results indicate that unintegrated HD increases in a chronically infected cell line after TNF-alpha induction. This phenomenon, which previously had been observed only with acute infections, may offer insight into basic pathogenic mechanisms.