In-vitro studies on the effect of temperature on the penetration of antimicrobial agents into the middle ear. The purpose of the study was to evaluate in-vitro the penetration of the antimicrobial agents used in myringotomy into the middle ear fluid. Experiments were carried out on four male Beagles. The eardrum was ruptured in all animals. A piece of gelatin sponge impregnated with 5% phenol or iodopovidone was placed on the eardrum for 5 min. The eardrum was then opened, and the middle ear fluid was aspirated for the next 10 min. The antimicrobial agents were tested in three different combinations (phenol-benzylpenicillin; iodopovidone-benzylpenicillin; iodopovidone-amoxicillin). The pH and the concentrations of benzylpenicillin and amoxicillin were measured in the middle ear fluid during this period. The concentration of benzylpenicillin increased in all cases, although not significantly. In contrast, the concentration of amoxicillin was always significantly higher in all cases when the material contained iodopovidone. The combination of phenol-benzylpenicillin yielded a low concentration of benzylpenicillin in the middle ear fluid, with no significant change over time. There was a slight increase in pH, without significant difference in any of the experimental groups. When iodopovidone was added, the concentration of amoxicillin increased, whereas the combination of phenol-amoxicillin gave rise to a significant decrease in the concentration of benzylpenicillin and an increase in the pH. All these changes are probably attributable to the basic characteristics of the antimicrobial agents used.