The presence of antibiotic residues in milk not only is a potential consumer risk but also may cause serious problems in the fermentation processes used in the dairy industry. There is very limited information available on the effect of heat treatments on aminoglycoside activity in milk. For this reason, the objective of this study was to analyze the effect of different heat treatments (60 degrees C for 30 min, 120 degrees C for 20 min, and 140 degrees C for 10 s) on milk samples spiked with four aminoglycosides (gentamicin, 50, 100, and 200 microg/liter; kanamycin, 300, 600, and 1200 microg/liter, neomycin, 200, 400, and 800 microg/liter; and streptomycin, 200, 400, and 800 microg/liter). The method used was a bioassay based on the inhibition of Bacillus subtilis BGA. Statistical analysis of the three heat treatments studied showed that the one at 60 degrees C for 30 min did not inactivate the aminoglycosides, the treatment at 140 degrees C for 10 s produced inactivation levels of between 17% for kanamycin and 40% for neomycin, and the classic sterilization (120 degrees C for 20 min) showed a high heat inactivation (>95%) for all the concentrations of aminoglycosides tested with respect to the samples without treatment (control group).