Rate-zonal centrifugation of a reduced and alkylated respiratory mucin preparation identified a protein-rich fraction. This was subjected to trypsin treatment and one of the many liberated peptides was purified and its N-terminal sequence determined. The peptide was identical to a 14 amino acid sequence from the scavenger receptor cysteine-rich domain containing glycoprotein gp-340. A polyclonal antiserum, raised against the peptide, stained the serous cells in the submucosal glands of human tracheal tissue. The glycoprotein was purified from respiratory mucus by density-gradient centrifugation, gel chromatography, and anion exchange chromatography. The molecule exhibited a heterogeneous distribution of buoyant density (1.28-1.46 g/ml) that overlapped with the gel-forming mucins, was included on Sepharose CL-2B and was quite highly anionic. SDS-PAGE indicated a mass greater than 208 kDa and measurements performed across the molecular size distribution indicated an average M(r) of 5 x 10(5) with a range of M(r) from 2 x 10(5) to 1 x 10(6). Gel chromatography of respiratory mucus extracts ("associative" and "dissociative") indicated that this glycoprotein forms complexes that may involve the large gel-forming mucins MUC5AC and MUC5B. Rate zonal centrifugation suggested such complexes are more likely to involve MUC5B rather than MUC5AC mucins.