A homogenous enzyme immunoassay for the quantitative determination of netilmicin in serum was developed. The procedure utilizes a commercially available assay for gentamicin (EMIT; Syva Co., Palo Alto, Calif.). The method was adapted to a microcentrifugal analyzer, and log-logit regression analysis was performed with a computer. The results of samples assayed by this method correlate well with microbioassay (r2=0.985) and radioimmunoassay (r2=0.986). This method is not only precise and accurate, but also very rapid and economical and compares favorably to other available methods of netilmicin assay.