Radioimmunoassays for unique aminoglycoside antibiotics, three fortimicin components, have been developed using antisera obtained from rabbits injected with fortimicin A-, isofortimicin A- or fortimicin B-BSA conjugate, respectively. Fortimicins were tritiated with N-succinimidyl-[2, 3-3H] propionate. For fortimicin A, the standard curve was linear to logit-log plot yielding an sensitivity of 0.2 ng/tube. A correlation coefficient of 0.99 was obtained between the radioimmunoassay and a microbioassay for fortimicin A in human sera. In the assay cross-reaction occurred with fortimicin derivatives and there was no cross-reaction with neomycin, kanamycin, sagamicin, amikacin, dibekacin, penicillins and cephalosporins. From the comparison of the specificity observed in three kinds of antisera, the following conclusions have been obtained: (1) Anti-fortimicin A and anti-isofortimicin A antisera were specific for the corresponding haptens. (2) In fortimicin B-BSA conjugate and 3H labeled antigen of fortimicin B, the fortamine ring might be inverted from the 4C1 confirmation to the 1C4 conformation. Thus the specificity of anti-fortimicin B antisera for fortimicins resembled to that of anti-fortimicin A antisera.