Biochemical composition of middle ear effusions (MEE) and serum was compared both in experimentally induced middle ear inflammation in squirrel monkeys and in otitis media in humans. The MEE and serum protein concentrations were similar in the animal experiments. In human MEE the total protein concentration of both serous and mucoid effusions was higher than the proteins of the serum. Higher concentrations of potassium and lower concentrations of glucose in human MEE than in serum were also observed. Activities of various oxidative (lactate dehydrogenase, malate dehydrogenase) and hydrolytic (leucine aminopeptidase, alkaline and acid phosphatase, and lysozyme) enzymes in MEE and serum were compared. The ratio of enzyme activity between MEE and serum (MEE/Serum) was greater than one in all enzymes studied. Mucoid MEE had higher activity of enzymes than serous effusions in general. Lactate dehydrogenase isoenzyme patterns were compared on electropherogram. Isoenzyme fractions 1 and 2 were each smaller in MEE than in serum whereas 4 and 5 had a significantly higher activity in MEE than in serum. Higher activities of enzymes in MEE as compared with serum are consistent with the hypothesis that MEE results from inflammatory processes occurring in the middle ear cavity. The enzymes of MEE seem to have multiple origins, namely, enzymes normally present in blood, enzymes from the inflamed middle ear mucosa, and enzymes from leucocytes present in effusions.