A newly developed radioimmunoassay was used to measure the concentration of vancomycin in 137 specimens of serum from patients being treated with this antibiotic. Of these sera, 84 were also analyzed with a microbiological assay technique for vancomycin. Duplicate determinations were done with each of the techniques. Individual values and averaged values for both methods were used for statistical analyses. The correlation coefficients between all possible combinations of radioimmunoassay and microbiological assay results for the 84 sera were ≥0.99 (P<<0.01). Values for the regression coefficients of radioimmunoassay results on microbiological assay results ranged from 0.98 ± 0.01 to 1.03 ± 0.01. The mean percent deviation of radioimmunoassay versus microbiological assay results was -1.56 ± 0.60. A one-way analysis of variance demonstrated that the use of different standard curves for each batch of specimens assayed by microbiological assay did not significantly influence the results (P = 0.07). The microbiological assay and the radioimmunoassay for measurement of serum vancomycin levels yielded essentially identical results.