A radioimmunoassay for vancomycin has been developed which uses rabbit antiserum induced by vancomycin-bovine serum albumin conjugates and vancomycin labeled with 3H or 125I. Using either isotope, the method is simple and reproducible and has a sensitivity of 4 or 0.04 ng/ml, depending on the tracer used. This is 200- to 20,000-fold improvement in sensitivity compared with the most sensitive bioassay. Drug levels in serum or urine samples from patients receiving vancomycin can be determined by this assay procedure without processing. The data obtained with 3H and 125I labels were in good agreement. Patients' plasma vancomycin concentrations determined by radioimmunoassay correlated well with those determined by bioassay when the drug was administered intravenously. However, after oral administration the drug could be detected only by radioimmunoassay. The antiserum was evaluated for cross-reactivity with a wide variety of antibiotics and cancer chemotherapeutic agents, and no significant interference was found.