H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.