Activation of transcription by the Moloney Murine Leukemia Virus (MLV) and Simian Virus (SV40) enhancers was compared by transfecting recombinants containing these enhancers in either mouse or human cell-lines, and analysing RNA 48 h later by quantitative S1 nuclease mapping. The enhancers share the following properties. They stimulate transcription in an orientation-independent manner from the same startsites on the natural heterologous conalbumin (+62 to -102) or SV40 early promoter elements as well as on substitute promoter elements. The enhancers are most efficient when they are located directly upstream from the conalbumin (+62 to -102) promoter element, but they still stimulate transcription when they are either immediately downstream from the promoter element, or further upstream. Increasing the distance by interposing DNA sequences between the enhancers and the conalbumin promoter fragment results in decreased activation. Both enhancers show some cell-line specificity for activation of transcription. However, in all cell-lines and constructions tested the MLV enhancer was always less efficient than the SV40 enhancer. These results suggest that the MLV and SV40 enhancers stimulate transcription by similar mechanisms.