Alternative splicing of the full-length, primary transcript into numerous subgenomic mRNAs is one way that lentiviral gene expression is regulated. Because the behaviors of different viral isolates might reflect in part differences in splicing, we investigated the patterns of splice site utilization by simian immunodeficiency viruses (SIVs) originally isolated from sooty mangabey monkeys (Cercocebus atys). We used reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, and DNA sequencing approaches to characterize SIVdeltaB670, a pathogenic and neurovirulent isolate, and SIVsmmH4, a related molecular clone. The majority of randomly selected SIVdeltaB670 and SIVsmmH4 partial cDNAs contained tat, rev, nef, and long terminal repeat (LTR) intron splice donor and acceptor sites positioned as expected based on the proviral sequence of SIVsmmH4. Nearly all (87%) of the partial cDNAs analyzed contained a spliced LTR intron. A greater number of partial cDNAs derived from SIVdeltaB670-infected cells contained putative alternatively spliced introns in comparison to SIVsmmH4, including two previously undocumented splice junctions involving the LTR intron splice donor. These data provide the first comprehensive analysis of splice site utilization by an isolate of SIV in comparison to a related molecular clone and the first characterization of SIVsmm splice site utilization.