Seminal and testicular characteristics were compared in 19 three-year-old and 23 two-year-old Quarter Horse stallions. Semen was collected, and testicular evaluations made on all stallions once in April or May and again 60 days later. Semen was collected from stallions twice, one hour apart, at each evaluation. Average testicular tone and scrotal width were greater in three-year-olds than in two-year-olds. Ejaculates of three-year-olds had higher motility scores (66% vs 47%) sperm movement (3.2 vs 2.4), normality (77 vs 71), and total number of normal, motile spermatozoa per ejaculate (4.3 × 109 vs 2.2 × 109). Two-year-olds had twice as many cytoplasmic droplets (11% vs 5%) as three-year-olds. Average sperm concentrations per ml of gel-free semen and gel-free volume of ejaculates were not different. Volume of ejaculate and motility of spermatozoa were positively correlated with pregnancy rate, whereas percent cytoplasmic droplets was negatively correlated with pregnancy rate in both groups of stallions. Pre- and post-ejaculatory urethral, seminal, and preputial cultures were examined for pathologic organisms on all stallions. Eighteen of 42 had positive cultures for Klebsiella spp., Pseudomonas spp., β-Strep spp., or E. coli. Nine two-year-olds and 3 three-year-olds had positive cultures for Klebsiella spp. All but one of these stallions were housed on wood shavings and allowed limited exercise. Three-year-old stallions had superior seminal quality as compared with two-year-olds.