We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated (3.5-\mathrm{kDa}) protein, based on the release of freesulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with (\left[{ }^{14} \mathrm{C}\right.) ]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with (1 %) KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the ({ }^{14} \mathrm{C})-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels ( (\sim 100 \mathrm{ng}) ) of SP-C and is not affected by surfactant protein (B) or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids ((\sim 30 %) at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein. o 1994 Academlc Press, Inc.