Hox genes are developmentally regulated in mammalian embryogenesis, according to temporally and spatially restricted patterns which are affected by retinoids, vitamin A derivatives which have a function as, or at least mimic the action of, axis-specifying morphogens. In the human embryonal carcinoma cell line NT2/D1, HOX gene clusters are activated by at least two retinoids, all-trans- and 9-cis-retinoic acid (RA), in a 3' to 5' sequential cascade which reproduces the activation pattern observed in early embryogenesis. We have studied the regulation of the early activated HOXD4 gene, which is expressed in human embryogenesis in multiple transcripts generated by the developmentally controlled use of alternative transcription start sites and polyadenylation signals. Transfection of a 2.9 kb HOXD4 upstream genomic region linked to a reporter gene in NT2/D1 cells, allowed the identification of two different promoters and a distal enhancer element necessary for RA-dependent gene activation. This element confers to a heterologous promoter the ability to be induced by RA in NT2/D1 cells, and transactivated by alpha, beta and gamma retinoic acid receptors (RARs), but not retinoid X receptor (RXR), in COS-7 cells. DNase I footprinting analysis allowed the identification of four sequences which bind nuclear factors from both RA-induced NT2/D1 cells and embryonic tissues with similar patterns. The use of specific antibodies allowed the identification of at least RAR beta in some of the DNA-protein complexes, although the four sequences bind single RARs transfected in COS cells much less efficiently, or not at all, when compared to a canonical RAR responsive element. Induction of the HOXD4 promoter-enhancer in the presence of a selective RAR alpha antagonist indicated that the RAR alpha-dependent RAR beta activation is nevertheless a necessary step in HOX gene activation. Our results indicate that the ligand-dependent activity of RARs upon specific, cis-acting regulatory elements may have a key role in the induction of early activated HOX genes in response to retinoids. However, RARs represent only a fraction of the transcription factors interacting with the RA-responsive HOXD4 enhancer, which appears to be a complex element requiring specific combinations of nuclear factors for its proper function.