Tobramycin serum levels as well as muscle and fat tissue content have been determined for 8 h in hourly intervals using an agar diffusion method following a single intramuscular injection of 2.5 mg/kg body weight to 29 children. In addition two specimens of spleen and one testis could be assayed for tobramycin content in these children. Tissue fluid was obtained by use of a Colworth Stomacher Nr. 80. At 1½ hours mean peak concentrations in serum were found to be 4.32 μg/ml, in muscle 0.93 μg/g and in fat 1.16 μg/g, from thereon declining to lower values. At 8 h tissue content fell below the smallest detectable amount of 0.01 μg/g tobramycin. At any time tobramycin content in fat (on average 24% of the corresponding serum level) was significantly higher than in muscle (17% of the serum level), whereas tobramycin content in spleen never exceeded that in muscle as well as in fat. Thus blood supply of the tissue does not seem to the main factor determining tobramycin content in various tissues.