We investigated the response to a panel of steroids of the mineralocorticoid receptor (MR) in Amur sturgeon and tropical gar, two basal ray-finned fish, whose ancestors evolved before zebrafish and other teleosts. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone were between 13 pM and 150 pM. Gar MR EC50s were between 8 pM and 55 pM. Such low EC50s support regulation of MR activity by these steroids in sturgeon and gar. Companion studies with human MR and zebrafish MR found higher EC50s compared to EC50s for sturgeon and gar MR, with EC50s for zebrafish MR closer to gar and sturgeon MR than was human MR. Zebrafish MR EC50s were between 75 pM and 740 pM; for human MR, EC50s were between 65 pM and 2 nM. Unexpectedly, progesterone was an agonist for all ray-finned fish MRs, in contrast to its antagonist activity for human MR. Sturgeon, gar and zebrafish MRs contain a serine corresponding to serine-810 in human MR, hypothesized as critical for progesterone antagonism. Progesterone is an agonist forSer810Leu MR. Our data suggests an alternative mechanism for progesterone as an MR agonist in these three ray-finned fishes.