To investigate the effects of copper and superoxide dismutase (SOD) content of seminal plasma on buffalo semen characteristics, 54 semen samples collected from buffalo bulls by a bovine artificial vagina were used. Semen characteristics (motility, viability, morphology, concentration and volume) were recorded. Seminal plasma was harvested by centrifugation and kept frozen until analysis. Seminal plasma copper content was determined by atomic absorption procedure and SOD was measured by using a kit. The mean total copper value of seminal plasma was recorded as 2.51 +/- 0.04 mg kg(-1) (Mean +/- SEM) and the mean total SOD values was 39.02 +/- 0.81 IU mL(-1). To reduce the range of variability, the data were categorized according to their motility records in 3 groups of Excellent (Ex, >90% motile, n = 33), Good (Go, 80-89% motile, n = 15) and Moderate (Mo, < 79% motile, n = 6). The mean motility, viability, copper and SOD values in Ex group was recorded as 92.24 +/- 0.51%, 94.00 +/- 0.48%, 2.56 +/- 0.04 mg kg(-1) and 39.52 +/- 0.57 IU mL(-1), respectively. These values were 81.66 +/- 0.62%, 85.26 +/- 0.95%, 2.38 +/- 0.11 mg kg(-1) and 36.48 +/- 1.51 IU mL(-1) in Go group and 71.66 +/- 1.05%, 77.00 +/- 2.94%, 2.55 +/- 0.10 mg kg(-1) and 50.66 +/- 2.51 in Mo group, respectively. The mean copper value in Ex group was highly (r = 0.600) correlated with SOD and correlated with sperm motility (r = 0.372) and viability (r = 0.363), while, in Go group it was highly correlated (r = 0.945) with SOD and sperm viability (r = 0.652) and in Mo group it was correlated (r = 0.874) with semen volume only. The mean SOD values in Ex group was highly correlated with sperm motility (r = 0.492) and viability (r = 0.490) and mean copper values, in Go group, it was highly correlated whit sperm viability (r = 0.659) and mean copper values and in Mo group it had no significant correlations with semen parameters. These results suggest that copper and SOD content of the buffalo seminal plasma have an influence on the sperm motility and viability which are the most important factors in semen fertility.