The long terminal repeats (LTRs) of cloned intracisternal A particles (IAPs) can function as effective promoters in heterologous and homologous cell types (K. K. Lueders, J. W. Fewell, E. L. Kuff, and T. Koch, Mol. Cell. Biol. 4:2128-2135, 1984) and respond to transcriptional factors induced by various nuclear oncogene products (S. Luria and M. Horowitz, J. Virol. 57:998-1003, 1986). Using the first 139 base pairs of the U3 region of a cloned mouse IAP LTR as probe, we demonstrated multiple exonuclease III stop sites which appeared specifically in the presence of nuclear extract protein. Various extracts gave similar footprints, but the amount of nuclear protein required varied up to 10-fold. Cell lines transformed with known nuclear oncogenes, such as adenovirus E1a and E1b (293 cells), simian virus 40 large T antigen (COS7 cells), and c-myc (MOPC-315 cells) had more and/or higher-affinity factors for the IAP LTR than extracts from HeLa, CV1, and NIH 3T3 cells did. DNase I footprinting revealed at least five distinct protein-binding domains within the 139-base-pair region. These domains correspond to segments of highly conserved nucleotide sequence among a number of IAP LTRs. Gel retardation studies with oligonucleotides encompassing the DNase I footprint sites showed that the nuclear factors are present in different proportions and different absolute levels in extracts from different cell types. Moreover, the oligonucleotide probes indicate that individual motifs can be occupied independently of one another. Three of the DNase I footprints include a sequence with homology to the simian virus 40 core enhancer and sequence motifs that closely resemble the binding sites for transcription factors SP1 and AP-1. The other two binding sites are not obviously related to previously recognized motifs. The multiple protein-binding sites in close proximity indicate the complex regulatory mechanism for IAP transcription.