Summary In a screening study, a common benzocaine-containing anesthetic was topically applied to the following species: dogs (n = 11), domestic shorthair cats (n = 38), Long-Evans rats (n = 22), Sprague-Dawley rats (n = 11), ferrets (n = 6), rhesus monkeys (n = 10), cynomolgus monkeys (n = 10), owl monkeys (n = 10), New Zealand White rabbits (n = 18), miniature pigs (n = 9), ICR mice (n = 4), C3H mice (n = 4), and C57BL/10SnJ mice (n = 24). All animals, except mice and rats, received a 2-second spray to the mucous membranes of the nasopharynx for an estimated dose of 56 mg. A 2-second spray to rodents' oral mucous membranes deliverd too great a volume of fluid for these animals; therefore, an equivalent dose was applied to the oral mucosa membranes by use of a 23-gauge needle and syringe. Initial (baseline) blood samples, as well as 4 blood samples taken every 15 minutes after drug application, were analyzed for methemoglobin (MHb), using an oximeter. Positive MHb response (> 3 sd above baseline) was seen in individuals of all groups. The study was repeated in dogs several months later to confirm low response. Response to benzocaine spray was observed in most animals tested, with response peaking between 15 and 30 minutes after dosing. Positive MHb response ranged from 3.5 to 38%, was detected in > 95% of individual animals, and ranged from 15 to 60 minutes after drug administration. Responses were variable because of the screening nature of the study and the topical route of drug administration, but the highest responses were observed in rabbits and cats, and the lowest were seen in mice and dogs. Methemoglobin could be a confounding variable for several types of studies; investigators should consider this toxicity of benzocaine-containing topical anesthetics and use appropriate alternative methods or drugs (ie, lidocaine).