The enzyme 5 alpha-reductase converts testosterone (T) to dihydrotestosterone (DHT). Although this enzyme has been localized to various regions of the pilosebaceous unit, its activity has not been studied in the follicular portion of either vellus or sebaceous follicles. The goal of our study was to determine the relative activities of 5 alpha-reductase within various regions of these follicles with particular emphasis on the infrainfundibulum. A finding of increased 5 alpha-reductase activity in upper follicles compared to epidermis might support the hypothesis that increased follicular production of DHT is involved in the hyperkeratinization observed in this region of the follicle in acne vulgaris. 5 alpha-reductase activity was determined at pH 5 (optimal for the type 2 isozyme) and pH 7 (optimal for the type 1 isozyme) in isolated infrainfundibular segments from sebaceous and vellus follicles, homogenized epidermis from various anatomical areas and in microdissected segments of the pilosebaceous unit from breast skin of normal subjects. Enzyme activity was also determined at pH 7 in cultured infrainfundibular keratinocytes and in interfollicular epidermal keratinocytes. Homogenates of infrainfundibular segments demonstrated significantly greater activity at pH 7 compared to pH 5 (P < 0.001), confirming activity of the type 1 5 alpha-reductase in this region. Activity of 5 alpha-reductase was much lower in homogenized epidermis and did not demonstrate a clear pH preference. Keratinocytes cultured from the infrainfundibulum demonstrated significantly greater 5 alpha-reductase activity compared to keratinocytes from interfollicular epidermis (P = 0.04). In the dissected segments of pilosebaceous units from breast skin, 5 alpha-reductase activity was greatest in the sebaceous gland followed by the sebaceous duct, infrainfundibulum, whole skin and epidermis. These data indicate that 5 alpha-reductase activity varies within regions of the pilosebaceous unit and compared with interfollicular epidermal cells, infrainfundibular keratinocytes have an increased capacity for producing androgens which may play a role in the follicular hyperkeratinization seen in acne.