Immunological properties of isepamicin (HAPA-B), a new semisynthetic aminoglycoside, were compared with those of gentamicin (GM). The results are summarized as follows. Rabbits were immunized by HAPA-B or GM with a strong adjuvant, Freund's complete adjuvant, and specific IgG antibodies were found in sera when assayed using PHA and PCA reactions. In sera from guinea pigs and mice immunized by HAPA-B or GM with aluminum hydroxide gel, no specific IgG nor IgE antibody was detected when assayed using PCA reactions. Specific antibodies (IgG, IgE) produced against HAPA-B and GM were confirmed in different animals immunized by chemically introduced antigens of HAPA-B-carrier protein conjugate and GM-carrier protein conjugate, respectively. Guinea pigs immunized by HAPA-B or HAPA-B-carrier protein conjugate with aluminum hydroxide gel did not exhibit typical systemic anaphylactic reactions when elicited with HAPA-B alone. HAPA-B had some immunocross-reactivity with GM. These results suggested that the antibody-producing activity of HAPA-B was similar to that of GM.