The striped pattern of fushi tarazu (ftz) expression found in the blastoderm of the Drosophila melanogaster embryo is generated largely through complex interactions between multiple transcription factors that bind to the zebra element of the ftz gene. A motif in the zebra element, the FTZ-F1 recognition element (F1RE), has been shown to bind a transcription factor, FTZ-F1 alpha, that is a member of the nuclear receptor family. We recently identified a second, related member of this family, FTZ-F1 beta, that also binds to this motif. To investigate the possibility that FTZ-F1 alpha and FTZ-F1 beta coregulate ftz transcription through the F1RE, we have studied the DNA binding properties of FTZ-F1 alpha and FTZ-F1 beta. We demonstrate that recombinant FTZ-F1 alpha and FTZ-F1 beta proteins produce similar in vitro DNase I footprint patterns on a 14-nucleotide region of the zebra element and bind to this site with similar affinities and sequence specificities. Using wild-type and N-terminally truncated receptors, we have determined that FTZ-F1 alpha and FTZ-F1 beta both bind as monomers to the 9-bp F1RE in the zebra element, as well as to an imperfect inverted F1RE repeat present in the Drosophila alcohol dehydrogenase gene. A polyclonal antibody raised against FTZ-F1 beta identifies a predominant F1RE-binding component in embryonic nuclear extracts. Although FTZ-F1 alpha is also present in these extracts, FTZ-F1 alpha and FTZ-F1 beta do not appear to form heterodimers with each other. Cotransfection assays in mammalian cell culture indicate that both receptors contribute to the net transcriptional activity of a reporter gene through their direct interaction with the F1RE. These data suggest that FTZ-F1 alpha and FTZ-F1 beta likely coregulate common target genes by competition for binding to a 9-bp recognition element.