Serum albumin from normal individuals and short-term hemodialysis patients was separated by isoelectric focusing into two bands with pIs of 4.7 (Alb-I) and 4.2 (Alb-II). However, serum albumin from long-term hemodialysis patients with CTS migrated as a single band with a pI of 4.2 (Alb-II). The major constituent of the 100,000 x g supernatant proteins from amyloid tissues that were collected from hemodialysis patients with CTS was Alb-II. Incubation of serum albumin by lysosomal enzymes in vitro caused the modification of Alb-I to Alb-II. The ratio of Alb-I/Alb-II was changed in vivo during the hemodialysis and in in-vitro experiments designed to circulate the sera. Lysosomal enzyme levels such as acid phosphatase, glucosidase, glucuronidase and dipeptidyl aminopeptidase in the serum of hemodialysis patients were higher than those of normal individuals. Plasma levels of acid phosphatase and cathepsin L were elevated during hemodialysis. These results suggest that the conformational change of serum albumin by proteolytic modification during hemodialysis may be involved in the hemodialysis-associated amyloidosis.