The western spotted skunk is a seasonally breeding mammal: most copulations occur in late September and early October. This study was performed to characterize the seasonal changes in concentrations of testosterone and in ejaculate quality. Captive males (n = 22) were maintained on a natural photoperiod for 15 months. Semen samples were collected by electroejaculation; testis size was measured; and blood samples were collected. Of 110 electroejaculation attempts, 104 (95%) resulted in successful fluid collection and 101 (97%) samples contained spermatozoa. Significant increases (P < 0.05) in serum testosterone concentration, testis size and ejaculate volume were observed from August to November. Mean concentration of testosterone in serum ranged from 0.15 ± 0.05 ng ml−1 in mid-January to 6.42 ± 1.79 ng ml−1 in early October. Mean testis size ranged from 1.22 ± 0.25 cm2 in February to 2.68 ± 0.08 cm2 in October. Mean ejaculate volume ranged from 11 ± 3 μl in March to 129 ± 22 μl in October. Seasonal changes in the number of spermatozoa per ejaculate or motility of spermatozoa were not observed. Mean number of spermatozoa per ejaculate was 8.14 ± 0.85 × 106 spermatozoa (n = 97); motility was 56 ± 2.4% (n = 93); semen pH was 7.76 ± 0.20 (n = 6); osmolarity was 394 ± 13 mmol kg−1 (n = 10); and 70.3 ± 1.5% of the spermatozoa were morphologically normal (n = 47). Concentration of testosterone in serum was highly correlated with testis size (r = 0.484, P < 0.001, n = 137), but none of the semen characteristics was found to be correlated with either concentration of testosterone in serum or testis size (P > 0.05). These data suggest that concentration of testosterone in serum, testis size and ejaculate volume increase seasonally as photoperiod decreases.