An automated system for HPLC-fluorometry of serum guanidino compounds was constructed. This system accomplished simultaneous removal of protein and uremic fluorescences, abundant in the sera of uremic patients, which interfere with the fluorometric assay. This system was applied to the detailed elucidation of the behavior of guanidinosuccinic acid and methylguanidine during and after hemodialysis therapy (HD). The uremic patients who are capable of excreting urine even under hemodialysis therapy showed low serum guanidinosuccinic acid and methylguanidine levels. The prolongation of the interval between HD for one of the patients capable of excreting urine was examined. The levels of guanidinosuccinic acid and methylguanidine did not significantly increase and no hazardous effect was observed by 2 d of prolongation.