Transient transfections of tissue culture cells with plasmids encoding the X protein of hepatitis B virus result in a transcriptional trans-activation of certain target genes. Our experiments reveal that several individual simian virus 40 enhancer elements and a control element present in the mouse major histocompatibility class I gene H-2Kb are able to mediate the trans-activating function of the X protein. The data suggest that known cellular transcription factors that bind specifically to the multiple enhancer elements participate in trans-activation.