Infection by the protozoan parasite, Toxoplasma gondii is widely prevalent in humans and animals throughout the world. Transmission takes place mainly by ingestion of raw or undercooked meat that contains parasite cysts or by ingestion of oocysts excreted in cat faeces, which can contaminate water and raw vegetables. The incidence of toxoplasmosis in urban areas can thus be also related to environmental contamination with oocysts. A direct measure of this environmental contamination by oocyst counting is unfeasible for technical reasons. An interesting alternative for measuring T. gondii urban spreading is the seroprevalence in free-living urban animals, used as sentinels, once they are exposed to similar risks of Toxoplasma infection-like humans. With this aim, we tested serum samples from stray cats and dogs for antibodies to T. gondii by indirect haemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 40% (40 of 100) of the cats, less than the 50.5% (101 of 200) found in dogs by ELISA (P < 0.05). Haemagglutination showed low resolution and concordance, precluding their use for diagnosis of T. gondii infection compared with ELISA. The prevalence of T. gondii was lower among stray cats probably due to their selective alimentary habits and lower water and food intake. Toxoplasma gondii seroprevalence in stray dogs and cats could be an indirect indicator of the parasite spreading in urban areas.