alpha 1-Cysteine proteinase inhibitor was isolated from normal and acute phase rat serum. The procedure, which includes successive fractionations on AcA 34, Cibacron blue Sepharose, DEAE-Sephacel, and hydroxyapatite, but avoids the use of an affinity chromatography step on a cysteine proteinase gel, led to the preparation of two electrophoretically distinct components. These resolved to a single band of apparent Mr = 68,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly higher Mr was estimated from gel filtration studies, possibly related to the rather high carbohydrate content (15.25% of the dry weight). The purified protein exhibited strong inhibiting capacity towards papain with a Ki of 5 X 10(-11) M. As shown by immunochemical quantitation as well as functional activity, a 6-10-fold increase in the alpha 1-cysteine cysteine proteinase inhibitor content was recorded in inflammatory serum, thus demonstrating the protein to be a typical acute phase reactant. Its partial physicochemical characterization (Mr, isoelectric point, extinction coefficient, amino acid and carbohydrate compositions) shows large similarities with alpha 1 acute phase globulin whose biological function remains unknown, and which is present at the same concentration (about 0.5 g/liter) in normal serum. Identity between the two molecules has been further demonstrated by double immunodiffusion analysis since a continuous precipitating line was observed when purified alpha 1-cysteine proteinase inhibitor was reacted against anti-alpha 1 acute phase globulin and anti-alpha 1-cysteine cysteine proteinase inhibitor antibodies.