The purpose of this study was to assess whether rat cystatin S, a cysteine proteinase inhibitor, is present in rat sebaceous glands, and to measure the effects of methotrexate on the expression of cystatin in these glands. With methotrexate treatment, the number of skin sebaceous cells expressing cystatin increased from 13.9% to 34.3% (P < .05). A smaller increase (from 15.3% to 23.9%; P = .1) was observed in Zymbal sebaceous glands. Type 2 cystatin could not be detected in the major salivary glands, nor in trachea, lung, stomach, small intestine, large intestine, spleen, liver, kidney, or pancreas, in any of the rats given either saline or methotrexate. Our results suggest that type 2 cystatin is a constituent of normal sebaceous glands, and that the amount of cystatin present in these glands increases with methotrexate administration. We speculate that, in addition to the protective functions ascribed to sebaceous lipids, sebum may augment the physical barrier of skin through secretion of cysteine proteinases that may be pharmacologically modulated.