Sequence variation plays a significant role in the pathogenesis and persistence of retroviral infections and is a major obstacle in the development of vaccines as well as therapies against lethal diseases caused by retroviruses. Recombination is one means by which sequence variation is generated. However, the basic molecular mechanisms of recombination are not adequately understood. In the present study, a spleen necrosis virus (SNV) recombination system was used to ask whether a known hot spot for mutation was also a hot spot for retroviral recombination. The system consisted of a pair of SNV vectors expressing two drug-resistance genes, constructed so that recombinants could be selected by a double resistant phenotype. Restriction enzyme site differences engineered into the vectors were used to map the location of recombination sites within relatively small intervals (55 to 420 bp). The vectors were modified to create two pairs that differed only by the presence of runs of identical nucleotides. The runs of identical nucleotides had been shown previously to be hot spots for frameshift mutations during SNV reverse transcription. Each vector pair was introduced into DSDh helper cells by infection. Viruses were harvested from doubly infected DSDh helper cells and used to infect D-17 target cells. Proviral sequences from 228 cell clones were analyzed by polymerase chain reaction and restriction enzyme digestion. Significant differences in the patterns of recombination were found between the two pairs of vectors. In particular, the frequency of recombination was higher than expected in the interval immediately following the runs. For both pairs of vectors, the overall pattern of recombination was nonrandom and one region was refractory toward recombination.