Human 17beta-hydroxysteroid dehydrogenase (17-HSD) type 1 predominantly catalyzes the 17beta-reduction of estrone to estradiol. The present results, however, show that rat 17-HSD type 1 equally uses both estrone and androstenedione as substrates. Analyzing the activity of various rat/human chimeric enzymes indicated that the region between amino acids 148 and 268 is responsible for the difference in substrate specificity, which is in line with the structural data showing that the recognition end of the active site is primarily at residues 185-230. The enzymes are highly conserved between amino acids 148-191, and the data indicate that in this region Asn152HisAsp153Glu and Pro187Ala variations are most closely related to the differential steroid specificity. The structural analyses furthermore suggested that the presence of His instead of Asn at position 152 of the human enzyme might result in considerable rearrangement of the loop located close to the beta-face of the A- and B-rings of the bound substrate, and that the Pro187Ala variation could modify the flexible region involved in substrate recognition and access of the substrate to the active site. Altogether, our results indicate that the Asn152His and Pro187Ala variations, together with several amino acid variations at the recognition end of the catalytic cleft built by residues 190-230, alter the structure of the active site of rat 17-HSD type 1 to one more favorable to an androgenic substrate.