Autism spectrum disorders (ASD) are a heterogenous group of neurodevelopmental disorders with a significant rate of increase over the last years. T. gondii is a ubiquitous intracellular pathogen affecting approximately 30-50% of human population, with special preference to the central nervous system (CNS). Accumulating evidence suggest that latent chronic toxoplasmosis play a role in triggering and development of many psychiatric and neurological disorders, but so far there is no clear epidemiological link with its prevalence in ASD. The aim of the study was therefore to estimate the seroprevalence of chronic toxoplasmosis among autistic children, determine the changes in serum levels of IFN-g and nitric oxide (NO) in T. gondii-positive and T. gondii-free patients, and evaluate the combined effect of both diseases on the Childhood Autism Rating Scale (CARS) score. Forty-six children with ASD (mean age 6.1 ± 2.2 years; 41 boys, 5 girls) were studied for anti-T. gondii IgG antibody seropositivity (ELISA kit, DRG Int), and their serum IFN-g (ELISA kit, Ray Biotech) and NO (one-step enzymatic assay) concentrations were measured. Chronic toxoplasmosis was found in 23.9% of the studied patients (8 males, 3 females), while among 50 age-matched control children (45 boys, 5 girls) 4% (2 boys) were T. gondii-positive (c2 = 8.11; P < 0.0043). Autistic children with toxoplasmosis had markedly increased both serum IFN-g and NO levels (11- and 5-fold median peak increases, respectively) compared with T. gondii- free participants. A statistically significant positive correlation was found between the serum IFN-g and NO levels (r = 0.79, P < 0.001). These molecular disturbances may exert harmful effects on further development of the CNS in the infected individuals, although no marked difference in the CARS score was detected, probably because of young age and immune function maturation of the participants. The obtained results strongly suggest that latent chronic T. gondii infection have an important impact on triggering and development of ASD, at least in a subset of autistic children, and this requires some modification(s) of its diagnostic procedures and treatment regimens.