The safety and pharmacokinetics of mupirocin, a novel antibiotic produced by Pseudomonas fluorescens, was assessed by a single application followed by 9 times repeated application to eight healthy male Japanese subjects. 0.2 g of calcium mupirocin 2% ointment was applied to anterior nares in each application. The study design was double-blind and placebo-controlled. In the study, no drug related symptoms were reported. In addition, no abnormalities were noted on physical examination, ECG recording or laboratory examination. Serum concentration of mupirocin was determined utilizing bioassay. Serum concentration of monic acid and urinary concentration of mupirocin and monic acid were determined utilizing HPLC. Both mupirocin and monic acid were less than lower limit to quantitation in serum and in urine. It was concluded that safety of calcium mupirocin 2% ointment was confirmed in healthy male Japanese subjects. It was also confirmed that even after repeated application, absorption of mupirocin was negligible.