The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and p I range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.