Roe deer (Capreolus capreolus) is an important game animal in Spain. Sera from 278 roe deer from eight areas in mainland Spain were assayed for antibodies to Toxoplasma gondii by modified agglutination test (MAT). Titers of 1:25 or higher were found in 109 (39.2%) of 278 deer. No significant differences in antibody prevalence were found between sex or age categories. In contrast, significant differences in seroprevalence between locations were evident. Roe deer from the Northern coastal habitats (high humidity and roe deer density) had the highest prevalence, compared with low prevalence in Central Spain (arid areas and low roe deer density). There was a positive correlation between antibody prevalence and mean annual rainfall (r(s)=0.85, n=8, P<0.01). These findings have environmental and/or public health implications because venison can be an important meat source of T. gondii infections for humans and feral cats.