Nucleotide sequence analysis of the cellular sequences flanking the integrated ecotropic (mouse-infectious) murine leukemia provirus of BALB/c mice indicated that the murine leukemia provirus is integrated in opposing transcriptional orientation within a solo long terminal repeat (LTR) of the VL30 family of endogenous retrovirus-related sequences. To quantify the effect of this integration event on the ability of the ecotropic provirus to be expressed, we constructed recombinant molecules that carried the chloramphenicol acetyltransferase (cat) gene and various viral LTRs and determined the CAT activity induced by these constructs after transfection of NIH 3T3 cells. Our results indicate that the BALB/c ecotropic LTR is about 10-fold more active than the VL30 LTR. The presence of the VL30 LTR did not affect the transcriptional activity of the ecotropic LTR in the context of the integration event. Our results also indicate that the LTRs of the BALB/c provirus are less transcriptionally active than are the proviral LTRs of AKR murine leukemia virus and the Harvey murine sarcoma virus.