Of the 920 cows tested, 56.7% showed antiretroviral serological reactivity. Prevalence rates (95% confidence interval) of antiretroviral antibodies among individual dairy cows in Ontario were: BIV 5.5% (4.0-7.0), BLV 25.7% (22.9-28.6), and BSV 39.6% (36.4-42.8). The following percentages of cows showed serological reactivity against the specified retroviruses: BIV 2.3%, BLV 14.0%, BSV 27.5%, BIV and BSV 1.3%, BIV and BLV 0.9%, BLV and BSV 9.9%, BIV and BLV and BSV 0.9%. These rates of sero-positivity are similar to those found in other countries. Serological test results were not adjusted for sensitivity and specificity. The prevalence rates of antibodies to the three retroviruses (BIV, BLV, and BSV) were significantly different, but no associations were observed between specific retroviral serological test results among individual cows. The prevalence rates of BIV and BSV seropositivity were constant across Ontario, whereas, there was a significant trend for the prevalence rate of BLV seropositivity to decrease going from southwestern to eastern Ontario; cows in eastern Ontario had approximately half the prevalence rate of those in southwestern Ontario. Cows that were seropositive for BSV were significantly older than BSV seronegative cows. There was no association between culling rate and BSV serology. Significant negative associations were found with winter or summer housing of calves separate from adults and summer outdoor exercise for dry cows. The use of calf hutches in the summer had a significant positive association with BSV seropositivity. Regression analyses were done to assess the association of retroviral (BIV, BLV, and BSV) seropositivity on calving interval, milk somatic cell count, and milk production. Serological test results for BIV, BLV, and BSV were entered into all models and all models were adjusted for intra-cluster (intraherd) correlation. Herd size and age were found to be important confounding variables. BIV seropositivity was not associated with any changes in production using this approach, however when considered in isolation BIV seropositivity remained associated with decreased milk production. BLV seropositivity was significantly associated with longer calving intervals and higher somatic cell counts in older cows. As well, in older cows, BSV seropositivity was significantly associated with higher milk production.