Using an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth microsomes and the Golgi subfractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against α₁-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with ¹²⁵I-protein A or ¹²⁵I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa α₁-protease inhibitor, which was indistinguishable from the serum α₁-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of α₁-protease inhibitor found in the microsomal fraction. Similar results were obtained for the β subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.