Background: Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups.Methodology/Principal Findings: Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4-68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R2 = 0.954, p,0.05). Conclusions/Significance: The present results could indicate a high prevalence of A. cantonensisin Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.