Trichophyton rubrum parasitizes the keratinized tissues of humans and proteinases secreted by this organism could function to allow this fungus to metabolize and invade host tissues. The effect of culture medium and glucose on the production of proteinases by cultures of T. rubrum was studied and the expression of these enzymes as a function of time in culture was followed. The fungus was grown to stationary-phase in Sabouraud's glucose broth and the original broth was then replaced with neopeptone, elastin salts, or keratin salts medium. The culture medium was assayed for azocollytic, elastinolytic and keratinolytic activity. Proteolytic activity was also analysed by electrophoresis of culture medium on gelatin and elastin substrate gels. T. rubrum expressed relatively high levels of azocoll-, elastin- and keratin-degrading activity in all of the types of media tested. Glucose partially repressed expression of proteolytic activity, the effect being dependent on the time and conditions of culture. Generally, expression of azocollytic activity peaked in the third week of culture, expression of elastinolytic activity peaked in the first or second week of culture, and expression of keratinolytic activity peaked at different times depending on the composition of the medium. These proteinases probably function to provide the fungus with a source of carbon, nitrogen, and sulfur and may also be responsible for the invasion of host tissues by T. rubrum in the rare cases of disseminated disease.