Guanidino compounds are increased in uremia and are highly suspected to be uremic toxins. The serum levels of 11 guanidino compounds and the influence of a single hemodialysis were evaluated in 30 steady-state uremic patients undergoing maintenance hemodialysis. Guanidino compound levels were detected using liquid cation exchange chromatography with a highly sensitive fluorescence detection method. Highly standardized dialysis procedures were performed. Before hemodialysis, high levels were found for guanidinosuccinic acid, N-α-acetylarginine, argininic acid, creatinine, γ-guanidinobutyric acid, guanidine and methylguanidine. Guanidinosuccinic acid reached levels associated with toxic effects in vitro. After hemodialysis, although lowered, guanidinosuccinic acid, creatinine, guanidine and methylguanidine were still markedly increased. No differences in the percent decrease, during a single hemodialysis, of the studied compounds were found using different membranes such as cellulose acetate, cuprophane and polyacrylonitrile membranes. Substantial differences, however, in the percent decrease of the different guanidino compounds were found, ranging from 25 ± 13% for arginine to 74 ± 7.5% for guanidinosuccinic acid. Data reported here show that guanidino compounds are raised in serum of uremic patients undergoing maintenance hemodialysis, before as well as after a single hemodialysis, while substantial differences in the percent decrease of the different guanidino compounds are found.