The use of sensitization indices for expressing allergenic skin reactions in guinea pigs is described. The method is convenient for comparing allergens and cross-reacting substances and permits the use of both irritating and nonirritating challenge concentrations of allergens. It also permits determination of both optimal reading time and challenge concentrations for each experiment. By this technique commercial neomycin complex, neamine (neomycin A), neomycin B, neomycin C, and streptomycin were found to be allergenic in guinea pigs via intradermal (id) and foot-pad (fp) immunizations. The immunizing emulsion consisted of an allergen and an adjuvant containing Mycobacterium butyricum (MB) or Mycobacterium tuberculosis H37Ra (Ra). The adjuvant MB was as effective as Ra by the id route, but inferior to Ra by the fp route. The cross-reactivity of neomycin C was generally greater than neomycin B in guinea pigs sensitized to neamine, neomycin B, neomycin C, or streptomycin. In guinea pigs sensitized to neomycin complex by repeated immunizations, neomycins A, B, and C were effective elicitors of skin reactions, whereas the N-acetylated derivatives of the components failed to cause reactions. This finding is interpreted to mean that the amino groups of the aminoglycosides are the coupling sites to host proteins in the processes of sensitization and elicitation of skin reactions in vivo.