Abstract The adsorption properties of Sephacryl S-200 are dependent upon the pH of the eluent buffer employed at the net surface charge of the solutes under investigation. At pH 3.5 the gel adsorbs acidic, neutral and basic proteins. By raising the pH of the eluent to 5.5 all adsorbed material can be desorbed from the gel. At pH 5.5 the gel showed no tendency to adsorb proteins of any surface charge. At this pH the gel acts as a passive molecular sieve. At pH 8 the gel begins to behave as a cation exchanger and strongly adsorbs any protein that is positively charged at this pH. Acidic or neutral proteins are eluted unretarded. These cation-exchange properties can be eliminated by including at least 0.2 M NaCl in the eluent buffer. At low ionic strength of the eluent buffer, DNA, tRNA and rRNA are adsorbed on to the gel at pH 3.5 or 5.5 but are completely desorbed by stepwise elution with 0.5 M NaCl in equilibrating buffer. The HETP values calculated for several solutes indicate that Sephacryl S-200 gives higher resolution and less zone-spreading than does Sephadex G-200.