An accurate and sensitive assay for vancomycin in serum and body fluids has not been available. This paper reports an assay for vancomycin that can detect serum and fluid levels as low as 0.8 mug/ml. A disk diffusion technique was designed employing buffered glucose minimal salts agar and Bacillus subtilis as an indicator strain. A linear relationship was obtained between zone diameter and concentration for vancomycin standards from 0.8 to 50 mug/ml prepared in pooled human serum. Results were accurate (<10% error) and reproducible (within-sample standard deviation, 0.25 mug/ml) for concentrations of from 0.8 to 25 mug of vancomycin per ml. Zone diameters were at least 6 mm larger on minimal salts agar than on standard assay media. The increased sensitivity and accuracy of the assay make it possible to accurately measure levels in cerebrospinal fluid and dialyzate fluid as well as in serum.