In the stallion, inconsistent sexual behavior and variable semen quality are common. This reproductive variability has been attributed to differences in circulating hormone concentrations. In order to further examine this relationship, 7 miniature stallions were observed for sexual behavior and semen characteristics. Blood was also drawn from each stallion 15 min before mating (time -15), immediately following ejaculation (time 0) and at times following ejaculation (times +15, +30, and +60). Plasma was later analyzed for concentrations of testosterone (T), estrone sulphate (ES) and cortisol. Semen was evaluated for volume, sperm concentration and progressive motility. Sexual behavior was quantified by assigning a libido score to each stallion, recording reaction time and the number of jumps required for ejaculation. Upon statistical analysis, data revealed both ES and cortisol increased at the time of semen collection (P < 0.05), while T did not. Regression analysis revealed that ES and the ratio of ES to T at times -15, +30, and +60 were negatively correlated to libido scores. Additionally, a positive relationship was found between ES at times -15 and +60 and reaction time, as well as between cortisol at times -15, 0, and +15 and libido scores. No relationship was observed between T and sexual behavior. However, T at time -15 was positively correlated to progressive motility, and the ratio of ES/T at time -15 was negatively correlated to progressive motility. No other association was detected between ejaculate parameters and hormone concentrations. These results not only serve to enhance understanding of stallion hormone profiles, but also provide further insight into the hormonal control of sexual behavior and sperm production. This knowledge can be used to generate improved management techniques for stallions that are inconsistent in sexual behavior and sperm output.