We examined more than 2,800 human and animal sera for antibodies to four serogroups of Legionella pneumophila by using the microagglutination test. Antibody titers of greater than or equal to 1:64 were considered positive. The occurrence of positive equine sera (31.4%) was significantly higher than the occurrence of positive sera in cattle (5.1%), swine (2.9%), sheep (1.9%), dogs (1.9%), goats (0.5%), wildlife (0%), and humans (0.4%). The highest titer measured in horses was 1:512. The occurrence of positive sera in horses was related directly to age. In horses less than or equal to 1, 2 to 3, 4 to 7, 8 to 12, and greater than or equal to 13 years old, the percentages of positive sera were 0, 10.1, 30.3, 44.9 and 58.1%, respectively. When we compared age-specific serogroup-specific rates in horses from Colorado and Pennsylvania, we found differences. With horses 8 to 12 and greater than or equal to 13 years old, there was a significantly higher (P less than 0.05) occurrence of sera that reacted to serogroups II and III in horses from Pennsylvania. Of 242 positive sera, 43.8% reacted to a single serogroup (serogroup III or I most commonly), and 56.2% reacted to multiple serogroups (serogroups II and III or serogroups I, II, and III most commonly). A high percentage of seropositive horses suggested that horses are commonly infected with L. pneumophila or related organisms, and the age-specific rates of occurrence indicated that infection was related directly to duration of exposure. A definitive demonstration of equine infection will depend on isolation of the agent and repetition of this serological study with antigens obtained from organisms isolated from horses.