A radioimmunoassay is described for the aminoglycoside antibiotic, gentamicin. Iodinated gentamicin was prepared using an 125I-labeled acylating agent: 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester which spontaneously reacts with gentamicin to produce an iodinated conjugate. The radioimmunoassay with iodinated gentamicin produced a straight line on a logit/log plot yielding an assay sensitivity of 80 pg. A Scatchard plot plus an equilibrium technique were used to estimate the affinity constant at 8 X 10(9) 1 mol-1 and 10.5 X 10(9) 1 mol-1, respectively. Kanamycin and neomycin, two chemically related aminoglycoside antibiotics, did not crossreact with gentamicin until concentrations were 10(5) greater than those found in blood. Assay of patients' serum samples by both a microbial assay and radioimmunoassay resulted in a statistically significant (p less than 0.001) correlation between the two methods.