The phylogenetic relationships of 56 nuclear hormone receptors from both invertebrates and vertebrates were determined by the parsimony method (PAUP). The consensus tree suggests that the ancestral gene diverged into five major subfamilies, each of which evolved into at least one cluster of related molecules. These subfamilies are represented by: (1) thyroid hormone receptors (TR); (ii) steroid receptors (SR); (iii) retinoic acid receptors (RAR), retinoid X receptors (RXR), and the chicken ovalbumin upstream promoter transcription factor 1 (COUP) group; (ix) peroxisome proliferator-activated receptors (PPAR); and (v) vitamin D receptor (VDR) and knirps (kni) group. Although the neighbor-joining (N-J) method clustered the receptors into a greater number of subfamilies, it was evident that the components of the terminal receptor subgroups were similar to those found in the PAUP tree. These terminal clusters might then represent phylogenetically stable relationships. The positions of some orphan receptors were perturbed when a different algorithm was employed in the analysis. Both PAUP and N-J evolutionary trees showed that the receptors within the subgroups of a major sublineage tend to recognize hormones of very similar structure. This finding suggests that the relative phylogenetic position of orphans to well-characterized receptors might be exploited to predict the type of ligand they would recognize.