Guanidine derivatives are suspected of contributing to the toxic manifestations of uremia. We describe a method for measurement of guanidine derivatives in 5-ml samples of plasma by liquid chromatography. Concentrations of guanidinosuccinate and guanidinobutyrate in plasma were significantly increased both in undialyzed patients with chronic renal failure (5.54 +/- 0.94 and 17.5 +/- 4.07 mg/liter) and those undergoing maintenance hemodialysis (2.35 +/- 0.41 and 19.4 +/- 3.99 mg/liter) when compared to healthy controls (less than 0.4 and 1.0 +/- 0.3 mg/liter, respectively). Creatine and guanidinoacetate concentrations tended to be higher in hemodialysis patients and lower in the undialyzed group of patients with chronic renal failure. This procedure provides a rapid, sensitive, and accurate method for the study of guanidine metabolism in persons with uremia.