Proteins absorbed directly onto nitrocellulose membranes were stained with amido black, ponceau S, colloidal silver, or Coomassie blue and solubilized in dimethyl sulfoxide and the absorbance was measured spectrophotometrically. The optimal wavelength of each dye/protein/nitrocellulose solution was found to be at 625, 529, 420, and 600 nm, respectively. A linear relationship was found between the protein concentration and absorbance at the appropriate wavelength for all the stains with individual purified proteins or protein mixtures. Protein (0.2-0.8 microgram) can be determined with the colloidal silver and 2-30 micrograms with the other stains. Coomassie blue produced variable background staining of the nitrocellulose and is therefore not recommended. Proteins transferred electrophoretically to nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel were also stained with the above dyes and solubilized in dimethyl sulfoxide. Amido black was the most sensitive stain, detecting proteins in the range of 1-10 micrograms. Components of the gel interfered with silver staining.