A short stretch of T-rich sequences immediately upstream of the polypurine tract (PPT) is highly conserved in the proviral genomes of human and simian immunodeficiency viruses (HIV and SIV). To investigate whether this 'U-box' influences SIVmac239 replication, we analyzed the properties of mutants with changes in this region of the viral genome. All mutants were either retarded in their growth (up to one month delay) or did not replicate detectably in CEMx174 cells. When U-box mutants did replicate detectably, compensatory changes were consistently observed in the viral genome. The most common compensatory change was the acquisition of thymidines immediately upstream of the PPT, but marked expansion in the length of the PPT was also observed. U-box mutants produced transiently by transfection were severely impaired in their ability to produce reverse transcripts in infectivity assays. Analysis of transiently produced mutant virus revealed no defect in RNA packaging or virus assembly. These results identify a new structural element important for an early step in the viral life cycle that includes reverse transcription.