Biological activities associated with colony-stimulating factor (CSF) from human leukemic urine were found to be selectively retained on an affinity adsorbent of Con A-Sepharose. Elution of activity was achieved using a linear gradient of eith alpha-methyl-D-mannopyranoside (alphaMM) or alpha-methyl-D-glucopyranoside (alphaMG), and resulted in significant increases in specific biological activity. Rechromatography of appropriate fractions indicated that retention of CSF activities was not artifactual. Pretreatment of the affinity matrix with alphaMM completely inhibited binding of CSF. Affinity chromatography of CSF on a Blue Dextran-Sepharose adsorbent was found to be an effective method for removing albumin, a major protein contaminant in urinary preparations. Treatment of CSF with neuraminidase had no effect on its in vitro activity; however, such treatment resulted in an increase in the isoelectric point of CSF from pH 3.5 to pH 4.7, as determined using both sucrose and polyacrylamide gel stabilized pH gradients. Relatively broad regions of biological activity were observed following isoelectric focusing of both neuraminidase-treated and untreated CSF, suggesting that activity was associated with a heterogeneous/polydisperse population of molecular species.