Mouse mammary tumor virus (MMTV) has frequently been used as an insertional mutagen to identify provirally activated mammary proto-oncogenes. To expedite and facilitate the process of cloning MMTV insertion sites, we have introduced a bacterial supF suppressor tRNA gene into the long terminal repeat (LTR) of MMTV, thus allowing selection of clones containing it in lambda vectors bearing amber mutations. The presence of supF in the LTR should circumvent the screening process for proviral insertion sites, since only those lambda clones with supF-containing proviral-cellular junction fragments should be able to form plaques on a lawn of wild-type Escherichia coli (i.e., lacking supF). The resulting virus (MMTVsupF) induced mammary tumors at the expected rate in infected mice, deleted the appropriate T-cell population by virtue of its superantigen gene, and stably retained the supF gene after passage via the milk to female offspring. To test the selective function of the system, size-selected DNA containing two proviral-cellular junction fragments from an MMTV supF-induced mammary tumor was ligated into gtWES.B, packaged, and plated on a supF-deficient bacterial host for selection of supF-containing clones. All plaques tested contained the desired cloned fragments, thus demonstrating the utility of this modified provirus for the rapid cloning of MMTV insertion sites.