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Department of Physiology, St George's Hospital Medical School, London.

1. We gave one of three radiolabelled precursors of mucus glycoconjugates ([3H]proline, [3H]glucose and [35S]sulphate) into the tracheas of anaesthetized cats for 3 h. In other cats [35S]sulphate was given by intravenous injection. 2. After a further 2 h, tracheas were removed and fixed. Serial actions were cut and alternate sections were stained with Haematoxylin and Eosin or prepared as unstained autoradiographs. Points on submucosal gland and surface epithelium were chosen with a grid on photomicrographs of the stained sections. Absorbance, which is proportional to autoradiographic grain density, was estimated on corresponding points on unstained autoradiographs by flying-spot microdensitometry. 3. With [3H]proline as precursor, the grain densities were greater over surface epithelium than over submucosal gland. With [3H]glucose, grain densities were greater over the surface epithelium in three cases, equal in one and greater over submucosal gland in the last. [35S]Sulphate, given either into the tracheal segment or intravenously, yielded grain densities that were greater over the submucosal glands than over surface epithelium. 4. The areas of submucosal gland the surface epithelium were estimated by point counting and the total content of radioactivity in the two structures estimated by multiplying mean absorbance by area. Ratios of the total radiolabel in surface epithelium to that in submucosal gland were consistently high when [3H]proline was the precursor and low with [35S]sulphate, given by either route. [3H]Glucose gave intermediate ratios. 5. Secretions washed from the trachea were subjected to gel-exclusion chromatography. Washings from tracheas labelled with [3H]proline contained some molecules eluting in the void volume of a Sepharose CL-4B column (suggesting a relative molecular mass of greater than 10(6) Da), but more of the radiolabel eluted in three peaks in the partially included volume. Density gradient ultracentrifugation of the void volume material gave radiolabelled peaks at densities of approximately 1.60 and 1.50 g ml-1, consistent with glycosylated proteins, as well as less dense material (less than 1.30 g ml-1), probably proteins with little or no glycosylation. 6. We discuss the justification of using these radiolabelled precursors to give relatively selective labelling of secretory products from submucosal gland and surface epithelium.