Amoxicillin is used as the drug of first choice in many situations in medicine and dentistry, in spite of several reports regarding bacterial resistance. There is little data about the tissue concentration of this antimicrobial agent. Serum levels of amoxicillin have been evaluated in detail, but tissue levels have not. This study was carried out to determine the tissue concentration of amoxicillin during the first 10 h after administration. Four polyurethane sponges were implanted in the backs of 54 male rats. After 14 days, they received 40 mg/kg of po amoxicillin suspension. The animals were killed in groups of 6 at 15, 30, 60, 90, 120, 240, 360, 480 and 600 min after the administration. Serum, placed on paper discs, and granulomatous tissue were assayed by a microbiological method using Mueller Hinton agar inoculated with 108 cfu of Staphylococcus aureus (ATCC 25923). After 18 h of incubation, the inhibition zones were measured. It was observed that the drug in the serum and the tissue reached higher concentrations than MIC and MBC within a period of 30 min and 8 h following administration. We conclude that this method can allow determination of antibiotic tissue concentration without the need for infecting the animal and, therefore, without the associated animal pain and suffering in presently used models.44197203