A bi-directional promoter, DP, was cloned by PCR amplification using the genomic DNA of melon as template. Analysis of its cis-acting elements in both directions revealed a series of inducible regulatory elements and some enhancer elements. To evaluate its transcriptional activity, DP in both directions was then cloned into vector pBI121 to replace the CaMV 355 promoter. DP in both directions also was inserted downstream of CaMV 35S to investigate whether the double promoter might affect expression of the uidA reporter gene at higher levels. Transient expression in cucumber leaves, stems, and fruits as well as in tobacco leaves and stems showed that DP in both directions drove transcription to much higher levels than did the single promoter CaMV 35S. However, activity of the double promoter was lower than the corresponding activity of the single promoter DP in both directions. These results demonstrate that DP is a natural bi-directional promoter, with much more activity than is found with the CaMV 35S promoter. Furthermore, in cucumber and tobacco, it is not suitable to insert DP in either direction downstream of the CaMV 35S promoter to form a double promoter.