Autoradiographic studies with tritiated amino acids have demonstrated that proteins of different composition might be synthesized in cells of the basal, spinous, and granular layers of the epidermis in man and newborn rats (1-3). The gradual morphological changes taking place during the process of epidermal keratinization were considered to result fundamentally from de novo synthesis of RNA and proteins in each of these three layers. Since Rudall (4) first demonstrated that different proteins were present at different levels in longitudinal sections of cow snout epidermis, numerous biochemical studies have detailed the isolation and characterization of soluble and insoluble epidermal proteins (4-12). Hoober and Bernstein (13) isolated HC104< soluble polypeptide (s) rich in histidine and glycine from newborn rats and suggested this substance was synthesized in the granular layer on the basis of prior autoradiographic observation (2). But the precise localization of these polypeptide (s) and other proteins isolated from epidermal cells remains unknown.No reliable methods are available for the separation of epidermal cell layers or organelles for biochemical studies. An isolation technic for keratohyalin granules was proposed by Ma-toltsy (14), but the amount isolated was insufficient for chemical analysis. Furthermore, in man it was found that about 10 per cent of granular cells located in the outermost layer were not synthesizing protein (3), indicating that protein synthesis evolves independently in cells. For these reasons conventional biochemical methods might prove inappropriate to yield precise information about the synthesis and metabolism of proteins in epidermal cells. Therefore, we have reinvestigated protein synthesis in the epidermis with concomitant cyto-logical observation.The present report concerns patterns of protein synthesis relative to keratinization in the epidermis of newborn rats as determined by autoradiographic technic after injection of isotopically labeled amino acids. Biopsies were taken sequentially to follow the movement of labeled cells into the cornified layer.MATERIALS AND METHODSNewborn rats of the Sprague-Dawley strain, 4-5 days old, and weighing about 7-8 grams were divided into groups of 10-15 each. They were injected intraperitoneally with 15 uC (in 0.05 ml of distilled water) of L-arginine-H3< monohydro-chloride (Spec. Act, 238 mC/mM), L-histidine-H3< monohydrochloride (Spec. Act. 3.42 C/mM), glycine-2-H3< (Spec. Act. 200 mC/mM), L-leucine-4.5-H3< (Spec. Act. 5.0 C/mM), L-lysine-H3< (Spec. Act. 1 C/mM), L-methionine-methyl-H3< (Spec. Act. 35.8 mC/mM), L-phenylalanine-H3< (Spec. Act. 4.32 C/mM), L-proline-3,4-H3< (Spec. Act. 5.0 C/mM), DL-serine-3-H3< (Spec. Act. 137 mC/ mM), L-tyrosine-3,5-H3< (Spec. Act. 3.41 C/mM), or DL-valine-3,4-H3< (Spec. Act. 137 mC/mM). The animals were sacrificed at hourly intervals for 6 hours, and at 12 and 24 hours after injection of the tracers. Skin samples were taken from the dorsal side of the animals and fixed in formol acetic acid alcohol. After dehydration, the samples were embedded in paraffin and cut at 4 microns. Auto-radiograms were made with Kodak Nuclear Track Emulsion, type NTB-2 and developed with Amidol developer followed by Edwal Quick Fixer after 10 and 21 days exposure. Sections were stained with hematoxylin and eosin. Since glycine is a precursor of