A high frequency of proviral acquisition has previously been reported in the offspring of SWR/J-RF/J hybrid mice. In the present study, it was investigated whether this proviral acquisition would be useful for large-scale insertional mutagenesis studies. A population of SWR/J-RF/J hybrid mice with a predominantly SWR/J background was created. Lines of mice with such a background and partially congenic for two active proviruses from the RF/J strain were generated (the insert lines). Control lines were derived from mice which had no proviral loci but had an otherwise similar genetic background. DNA samples of mice in the insert lines were screened for the appearance of new proviral loci by Southern hybridization. The rate of proviral acquisition, calculated from the observed number of new proviral loci was 0.023 new proviruses per mouse. This rate is lower than found in previous studies and too low for large-scale insertional mutagenesis studies. A sensitivity experiment indicated that there was adequate detection of new proviral loci. The number of segregating proviruses was consistent with the number of newly acquired proviruses actually detected. Two additional crosses between mice in the insert lines and SWR/J mice were performed. The rate of proviral acquisition was greatly increased when SWR/J females were initially mated to insert mice, but remained unchanged when SWR/J males were used. This suggested that mice in the insert lines had acquired a maternally transmitted factor, which was suppressing viral expression and thus reducing the rate of proviral acquisition.