The chemical characteristics of sulfation factor (PSF) and thymidine factor (PTF) have been studied in whole acromegalic plasma and in acid ethanol (AE) extracts of plasma. In whole plasma PSF and PTF are excluded by the XM-100 ultrafiltration membrane and behave as large proteins. After extraction with acid ethanol the mol wt appeared to be >3900≦12,400, suggesting that in native plasma PSF and PTF are either aggregated or bound to a larger carrier protein. Two species of peptides with PSF and PTF activity were found. Chromatography of whole plasma on DEAE-cellulose revealed a major peak of biologic activity which was not adsorbed at pH 8.5 and a smaller component which was adsorbed at pH 8.5 but eluted at pH 5.5. Electrofocusing of AE extracts revealed a sharp minor peak at pH 5.2 and a broader major peak at pH 6.6–6.7. The possibility that the more acidic component may be due to insulin cannot be excluded. HV electrophoresis of a purified AE extract revealed activity only in the neutral zone. Chromatography of an AE extract on CMC-cellulose at pH 5.6 in a linear ionic gradient between .01 and 0.40m resulted in the recovery of PSF and PTF in a small protein peak well separated from the bulk of other proteins. Rechromatography of the CMC eluate on Sephadex G-50 yielded a preparation with an increase in specific activity over native plasma of 6200× for PSF and 15,000× for PTF. This discrepancy probably reflects the removal of inhibitors having a greater effect on thymidine incorporation, since no separation of PSF from PTF was observed in any system. If a provisional molecular weight of 8000 is assumed and no allowance is made for residual contaminants, the purest preparation is highly active at 3.1×10−8 molar concentration.