A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an I?, greater than 0.50. We found that 1.25 wg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M, cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenie exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).