Recent reports of simian virus 40 (SV40) sequences in human tumors have prompted investigations into the poorly understood association of this polyomavirus with its primate host, the rhesus monkey (Macaca mulatta). In the present study we have used PCR to analyze tissues from 20 monkeys for the presence of SV40. Five of the animals, which were infected with simian immunodeficiency virus (SIV), were found to exhibit SV40-induced lesions and to have SV40 sequences present in their kidney and brain. Lesions associated with SV40 were not observed in 15 SIV monkeys, and SV40 DNA was detected in kidney and urine of only one of these animals. Three regions of SV40 DNA were examined in each tissue: the non-coding transcriptional control region (TCR), the sequences encoding the host range domain (HRD) within the carboxy-terminus of T antigen (TAg), and a portion of the VP1 gene. Each region contained nucleotide alterations compared to the SV40 reference strain 776. In all six animals, the TCR had an archetype structure containing a single 72 bp enhancer element. In addition, the TCR amplified from two animals lacked one of three copies of a GC-rich 21 bp repeat which is part of the promoter in strain 776. Multiple clones of unique HRD sequences were derived from different animals, and in some instances from the same animal. No correlation was found between a particular HRD sequence and its presence in a specific tissue type. Nucleotide changes identified within the VP1 gene indicate that this region, as with the closely-related human polyomavirus JCV, may permit the typing of the virus into individual strains. This study is the first to characterize SV40 sequences present in both healthy and SIV-infected animals and supports the suggestion that strain 776 is not the predominant type of SV40 circulating in its natural host.