Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.