In commercial production systems, the full expression of an animal's genetic potential is limited by its intrinsic and extrinsic environment. It is therefore necessary to include robustness as a breeding goal, because robustness is defined as the ability of an animal to express a high production potential in a wide variety of environmental conditions. The ability of mammals to produce sufficient cortisol on stimulation of the hypothalamic-pituitary-adrenal (HPA) axis is vital in its adaptation to stress. The biosynthesis of cortisol is dependent on the enzymatic activity of the microsomal enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17). Two isoforms for sheep (Ovis aries) CYP17, previously identified in two independent studies, differ by two nucleotides resulting in two amino acid differences (Ser210Gly and Tyr464Asn). The present study investigates the effect of these differences on cortisol production, as a function of the HPA axis activity, by comparing the catalytic activities of these isoforms. The activities of the CYP17 isoforms were compared by expressing the enzymes in COS-1 cells. The kinetic constants, Vmax and Km, which were determined for pregnenolone and progesterone (in the absence of cytochrome b(5)), showed no significant difference (P > 0.05) between the CYP17 isoforms. In contrast, a time course of the metabolism of pregnenolone, 17-hydroxypregnenolone and progesterone, assayed in the presence and absence of ovine cytochrome b(5) over-expression, showed significant differences (P < 0.05) between the isoforms. Wild type 1 CYP17 (WT1, GenBank ID: L40335) yielded more cortisol precursors than Wild type 2 (WT2, GenBank ID: AF251388). Site-directed mutagenesis indicated that a tyrosine residue at position 464 of WT1 increased the 17α-hydroxylation of progesterone compared to an asparagine residue at that position of WT2. In a subsequent insulin-induced hypoglycaemic stress test, the presence of WT1 resulted in a higher cortisol output from the sheep adrenal than the presence of WT2, as homozygous WT1/WT1 sheep produced more cortisol than heterozygous WT1/WT2 sheep. The SNPs located within the WT1 allele may therefore have a potential application in marker-assisted selection of sheep exhibiting a higher release of cortisol from the adrenal gland in response to stressors.