Two mechanisms of potential biologic antagonism of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis were studied: reduction of activity by ions and antibiotic binding. Antagonism by ions was assessed by examination of the activity of gentamicin against Pseudomonas aeruginosa in dialysates of serum or sputum in ion-depleted broth. The ionic content of the dialysates increased and reflected differences in the ion content of serum and sputum. Gentamicin had significantly less activity against P aeruginosa in sputum or serum dialysates than in ion-depleted broth alone. When gentamicin was mixed with serum or sputum before dialysis, the level of antipseudomonas activity of the sputum dialysates was significantly lower than that of the serum dialysate; this finding was correlated with greater binding by sputum. Thus, both binding and antagonism by ions evidently reduce the level of bioactivity of gentamicin in serum and in sputum. Purulent sputum, whether from children with cystic fibrosis or adults which bronchiectasis, is more inhibitory than serum; the greater degree of binding, rather than differences in the composition or quantity of cations, explains this difference.