The limulus endotoxin assay has been previously demonstrated to be the most sensitive method available for detection of bacterial endotoxin. A commercially available form of limulus amoebocyte lysate was used in this study for detection of Gram-negative corneal infections in both experimental animals and in a group of nine patients. The limulus assay enabled rapid detection of Gram-negative infections in both the experimentally induced ulcers in rabbits and in the patients studied. False-positive reactions did not occur in corneal infections due to either Gram-positive bacteria, fungi, or herpes simplex keratitis. The limulus test proved to be more sensitive than examination of Gram-stained smears of corneal scrapings and became positive earlier than bacterial cultures. The limulus test was helpful in the diagnosis of partially antibiotic-treated corneal infections but could not be used to assess the response to antimicrobial therapy, since endotoxin persisted in the corneal scrapings for some time after initiation of therapy.