Transcriptional enhancer sequences within the long terminal repeats (LTRs) of murine leukemia viruses are the primary genetic determinants of the tissue specificity and potency of the oncogenic potential of these retroviruses. SL3-3 (SL3) is a murine leukemia virus that induces T-cell lymphomas. The LTR enhancer of this virus contains two binding sites for the transcription factor CBF (also called AML1 and PEBP2) that flank binding sites for c-Myb and the Ets family of factors. Using cotransfection assays in P19 cells, we report here that CBF and c-Myb cooperatively stimulate transcription from the SL3 LTR. By itself, c-Myb had no stimulatory effect on transcription. However, when cotransfected with a cDNA encoding one form of the alpha subunit of CBF called CBFalpha2-451, a level of transactivation higher than that seen with CBFalpha2-451 alone was detected. The negative regulatory domain near the carboxyl terminus of c-Myb did not affect this activity. Electrophoretic mobility shift assays indicated that CBF and c-Myb bind to DNA independently. Therefore, it appears that the cooperative stimulation of transcription by these factors occurs at a step in the process of transcription after the two factors are bound to the enhancer. Sequences near the carboxyl terminus of CBFalpha2-451 were important for cooperativity with c-Myb, consistent with previous reports that this region contains an activation domain. However, CBFalpha2-451 failed to activate transcription from a version of the SL3 LTR in which the enhancer was replaced with five tandem CBF-binding sites. Thus, it appears that transcriptional activation of the SL3 enhancer by CBF requires that an appropriate heterologous transcription factor be bound to a neighboring site in the regulatory sequences.