Assays for gentamicin were performed on the sera of 67 patients by two methods for comparison of their utility. An enzymatic method that measures the adenylation of gentamicin by a bacterial extract and a standard agar diffusion technique with Bacillus globigii were selected. The coefficients of variation were 3% and 10%, respectively, and both methods were accurate at the 5% level. The methods correlated well (r = 0.96), although the results of the microbiological assay were, on the average, 18% higher than the results of the enzymatic assay. The enzymatic assay offered advantages of specificity and rapidity. The enzymatic assay is a dependable method that can be used in a centralized service for testing of a large number of sera on a fixed schedule.