Alternaria is a saprophytic fungus that is widespread in the environment; it is an opportunistic pathogen and causes disease in human beings and domestic animals. Fungal spores gain entry to the host through skin lesions and cause slow-growing, soft to firm, subcutaneous swellings, either with or without ulcers. An indirect ELISA was developed for the detection of anti-Alternaria immunoglobulin G (IgG) antibodies in serum to determine the prevalence of Alternaria exposure in domestic cats. Fifty-two of 63 cats had detectable levels of anti-Alternaria IgG antibody. There were no correlations between the concentration of antibody and the sex, breed or living environment of the cats, but cats less than two years of age had significantly lower concentrations than older cats. The cats with disease caused by culture-confirmed Alternaria infections did not have significantly higher concentrations of antibody than the healthy cats or cats with other diseases.