The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix–loop–helix heterodimer E47/BETA2 (β-cell E box transactivator 2; referred to here as β2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and β2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/β2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/β2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 β-cells. In the islet glucagonoma cell line αTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5–3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and β2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and β2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in αTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.