The radioenzymatic technique using aminoglycoside-6'-acetyltransferase is suitable for determining serum concentrations of aminoglycoside antibiotics that have a 6'-amino group. Standard curves constructed for each drug in normal human serum are generally satisfactory, but we encountered a pool of human serum (designated IN pool) that inhibited the acetylating activity of the assay. Standard curves for amikacin, kanamycin, gentamicin, and tobramycin in the acetylating assay were all abnormal with standards prepared in the IN pool. Acetylating activity was also inhibited with amikacin standards prepared in a filtrate of this serum pool. Heat inactivation (56 degrees C for 30 min) of the IN pool did not eliminate the problem. When the IN pool was diluted 1:10 in normal human serum, the standard curve obtained with amikacin was comparable to the curve with standards prepared in normal human serum. With amikacin standards prepared in a 1:2 dilution of the IN pool in normal human serum, an intermediate level of acetylating activity was observed. When this IN pool was used for the preparation of gentamicin and amikacin standards in a bioassay with Klebsiella pneumoniae ATCC 27799 as the test strain, valid standard curves and concentrations were obtained. Furthermore, no differences in the biological activity of amikacin were observed with Pseudomonas aeruginosa or Bacillus subtilis as the test strain. Excellent agreement between the microbial assay and the enzymatic assay was obtained with serum specimens tested for gentamicin (r = 0.89), tobramycin (r = 0.96), and amikacin (r = 0.96). The results obtained with the IN pool illustrate the need for regular use of check samples of known performance, independent from the standards, when determining antibiotic levels in serum.