The binding of nuclear proteins and the functional activity of the HIV-LTR enhancer repeats in different cell lines (Jurkat, CEM, H9, U937, Raji, B cells, T47D, HeLa, 293, and HepG2 cells) was investigated in vitro. Five distinct complexes formed with the enhancer repeat have been identified by an electrophoretic mobility shift assay. The distribution of these complexes varied qualitatively and quantitatively between nuclear proteins from different sources. In the extracts tested, transcription of the HIV-LTR 5' deletion mutants (-453/80, -176/80, -117/80, -103/80, -65/80, and -48/80) was initiated correctly. Transcriptional stimulation dependent upon the presence of the enhancer repeat sequences was observed in all nuclear extracts and was highest in Jurkat, Raji, and B cell extracts. The presence of specific factors and the functional activity of the enhancer repeats as well as other regulatory units in a variety of cells indicates limited host-cell restriction of HIV transcription initiation in vitro.