We previously demonstrated that a approximately 1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13-like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used beta-globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13-like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13-like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.