[Show abstract][Hide abstract] ABSTRACT: Lyme disease is a perfect model of the complex relationship between host, vector, and the vector-borne bacteria. Both dogs and horses in Romania are exposed to infection. The aim of the present study was to assess the seroreactivity against Borrelia burgdorferi sensu lato in dogs and horses from different regions of Romania. 276 samples from dogs and 260 samples from horses located in different regions of Romania were analyzed by ELISA and IFA, respectively. The effect of several factors potentially affecting seroreactivity (location, age, gender, occupation, and vector exposition risk) was evaluated using Fisher's exact test (R 2.12.0). The overall prevalence of anti-Borrelia antibodies was 6.52% (18/276) in dogs, with a significantly higher positivity (46.15%, 6/13, p = 0.0005) recorded in a midcountry region. Seroreactivity was correlated with occupation, with working dogs being more exposed. The results may indicate that Lyme borreliosis foci are restricted to small areas, but further studies on Borrelia prevalence in tick populations are needed to confirm this hypothesis. In horses, a global seroprevalence of 11.92% (31/260) was observed. No correlations were found between positive results and age, sex, county, or occupation. This is the first serological survey on antibodies to B. burgdorferi sensu lato in Romanian dogs and horses.