The hundred twenty-four isolates of anaerobic and microaerophilis organisms were examined with a polyvalent direct immunofluorescent antibody (DFA) reagent directed against Legionella pneumophila, serogroups (SG) I-IV. Three of 53 isolates of Bacteroides fragilis cross-reated with the SG I component of the reagent. Rabbit antisera to three SG I strains of L. pneumophila cross-reacted with these three B. fragilis isolates, and cross-reactivity was not due to preexisting rabbit antibodies; antisera to a fourth SG I strain did not cross-react with these isolates. Indirect fluorescent antibody (IFA) examinations of convalescent-phase sera from eight of 41 patients with B. fragilis group infections showed titers of greater than or equal to 1:128 or a fourfold or greater seroconversion against L. pneumophila, SG I. All eight patients had bacteremia due to B. fragilis, but five B. fragilis isolates available from these patients were DFA-negative. Thus, false-positive reactions may result from DFA as well as IFA examinations of sera from patients with B. fragilis infections.