The enhancer of Moloney murine leukemia virus (Mo-MuLV) contains an array of transcriptional control elements that direct viral gene expression in diverse cell types. The murine transcription factor Ets-1 was shown to bind to the LVb and LVc elements of the enhancer by DNase I protection and methylation interference assays. Enhancers containing disrupted Ets-1 binding sites were tested in transient expression assays in the murine T-cell line EL4.E1; alterations in the LVb element affected constitutive enhancer activity, while mutation of either the LVb or LVc element disrupted phorbol ester-induced enhancer activity. Members of the ets gene family of proteins display similar DNA-binding properties; therefore, we speculated that ets proteins other than Ets-1 also might bind these elements. Crude nuclear extracts of EL4.E1 cells were assayed to identify the protein(s) that potentially functions at the LVb element. The predominant binding activity was not Ets-1 but rather two independent DNA-protein complexes that comigrated in mobility shift assays. UV cross-linking and denaturing gel electrophoresis sized the two DNA-binding species, which we denoted p55 and p100. Immunoprecipitation combined with UV cross-linking identified p55 as the alpha subunit of GA-binding protein. The DNA-binding properties of p100 and several ets proteins were compared. Similarities suggested that p100 is also an ETS domain protein, possibly Elf-1. This strategy could be used to identify other ETS domain proteins in crude nuclear extracts. These findings suggest multiple ETS domain proteins could regulate gene expression of Mo-MuLV.