Since guanidinoacetic acid (GAA), a precursor of creatine, is synthesized mainly in the proximal tubule of the kidney where gentamicin (GM) nephrotoxicity often occurs, GM-induced renal cell damage was investigated using GAA synthesis in tubular suspension as an indicator. Results obtained were as follows: (1) GAA synthesis was significantly suppressed with 1 mM GM; (2) GM-induced decrease in GAA synthesis was recognized during incubation longer than 15 min; (3) furosemide significantly enhanced the suppression of GAA synthesis by GM. The results obtained parallel with those in the whole animal thus making GAA synthesis in tubular suspension a valid system for evaluating the GM-induced proximal tubular damage.