Expression of vertebrate Hox genes is regulated by retinoids such as retinoic acid (RA) in cell culture and in early embryonic development. Retinoic acid response elements (RAREs) have been identified in Hox gene regulatory regions, suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. Previously, two RAREs located 3' of the murine Hoxb1 gene, a DR(2) RARE and a DR(5) RARE, have been shown to regulate Hoxb1 mRNA expression in the neural epithelium and the foregut region, respectively; the foregut develops into the esophagus, liver, pancreas, lungs, and stomach. We have now examined the functional roles of these two types of 3' RAREs in regulating Hoxb1 expression at different stages of gestation, from embryonic day 7.5 to 13.5, in transgenic mice carrying specific RARE mutations. We demonstrate that the DR(5) RARE is required for the regulation of Hoxb-1 transgene region-specific expression in the gut and extraembryonic tissues, as well as for the RA-induced anteriorization of Hoxb-1 transgene expression in the gut. In contrast, expression of the Hoxb1 transgene in the neural epithelium requires only the DR(2) RARE. By in situ hybridization, we have identified a new site of Hoxb1 expression in the developing forelimbs at approximately day 12.5, and we show that, in transgenic embryos, expression in the forelimb buds requires that either the DR(2) or the DR(5) RARE is functional. Attainment of a high level of Hoxb1 transgene expression in other regions, such as in rhombomere 4 (r4) and in the somites, requires that both the DR(2) and DR(5) RAREs are functional. In addition, our transgenic data indicate that the Hoxb1 gene is expressed in other tissues such as the hernia gut, genital eminence, and lung. Our analysis shows that endogenous retinoids act through individual DR(2) and DR(5) RAREs to regulate Hoxb1 expression in different regions of the embryo and that functional redundancy between these DR(2) and DR(5) RAREs does not exist with respect to neural epithelium and the gut Hoxb1 expression.