Physiologic concentrations of calcium in serum antagonize the activities in vitro of gentamicin and tobramycin on Pseudomonas aeruginosa. The relative antagonism of serum to these antibiotics in vitro was determined on 21 strains of Pseudomonas, representing three each of the seven recognized immunotypes. Serum was more antagonistic to gentamicin than to tobramycin for 14 strains. Serum had little antagonism to either antibiotic for four strains. Serum was highly antagonistic to both antibiotics for three strains. In no instance was serum more antagonistic to tobramycin than to gentamicin. These results suggest that tobramycin may be more active against most strains in vivo than gentamicin at equal concentrations. The relevance in vivo of these in vitro findings remains to be determined.