Retroviruses contain two complete viral genomic RNAs in each virion. A system to study in a single round of replication the products of virions with two different genomic RNAs was established. A spleen necrosis virus-based splicing vector containing both the neomycin-resistance gene (neo) and the hygromycin B phosphotransferase gene (hygro) was used. Two frameshift mutants were derived from this vector such that the neo and the hygro genes were inactivated in separate vectors. Thus, each vector confers resistance to only one selection. The vectors with frameshift mutations were separately propagated and were pooled to infect DSDh helper cells. Doubly resistant cell clones were isolated, and viruses produced from these clones were used to infect D17 cells. This protocol allowed virions containing two different genomic RNAs (heterozygotes) to complete one round of retroviral replication. The molecular nature of progeny that conferred resistance to single or double selection and their ratio were determined. Our data demonstrate that each infectious heterozygous virion produces only one provirus. The rate of retroviral recombination is approximately 2% per kilobase per replication cycle. Recombinant proviruses are progeny of heterozygous virions.