Three fractions of choline O-acetyltransferase (ChAT) (EC 2.3.1.6) were solubilized from a nerve ending fraction of rat forebrain using three sequential washes of an increasingly chaotrophic nature (100 mM sodium phosphate, pH 7.4; 500 mM NaCl; 2% Triton DN-65) as previously described (Benishin, C.G., and P.T. Carroll (1983) J. Neurochem. 41: 1030-1039). The molecular weights of the soluble (NaP) and membrane-bound fractions (NaCl and 2% Triton DN-65) of ChAT, following partial purification, were determined using either gel filtration on Sephadex G-200, G-100 Superfine, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by "Western blotting" and immunochemical visualization of ChAT with four different anti-ChAT monoclonal antibodies (Ab8, Ab9, 4D7, and 1E6). Results obtained with gel filtration indicated that the NaP- and Triton DN-65-solubilized fractions of ChAT had molecular weights in the range of 73,000 to 78,000, whereas the NaCl-solubilized fraction of ChAT had a molecular weight in the range of 230,000 to 240,000. Results obtained with SDS-PAGE and Western blotting indicated that all three fractions of ChAT were composed of the same nonidentical subunits.