The XY Sex Reversal Syndrome of the horse is a condition associated with female or intersexual development in genetic males. In our previous study, 38 sex reversed XY mares were classified according to behavior, gross clinical phenotype, gonadal status, and H-Y phenotype. Four classes were described, ranging from potentially fertile female (Class I) to virilized intersex (Class IV). In the present study, serum testosterone concentrations were measured in 29 sex-reversed XY mares, 3 normal mares and 3 normal stallions. Serums were obtained during the breeding season (March-August), and were stored at -70 C until assayed. Serum testosterone concentrations in the normal XX mares ranged from nondetectable to 0.41 ng/ml; in normal XY stallions, from 1.04 ng/ml to 2.4 ng/ml; and in XY mares, from nondetectable to 5.4 ng/ml. Sex reversed mares previously assigned to Class I or II had serum testosterone concentrations ranging from nondetectable to 0.22 ng/ml. Serum testosterone concentrations in XY mares were correlated with sex phenotype and behavior. Although the range of steroid concentrations among XY mares may be quantified more accurately with increased sampling, serum testosterone concentrations can be used currently as an added parameter for study of the sex reversed condition.