Alpha-smooth muscle actin (ASMA) is an actin isoform present in the filaments of smooth muscle cells, myofibroblasts, and a specific region of hair follicle dermal sheath in vivo. We employed double immunofluorescence, two-dimensional electrophoresis, Western blots and DNA, protein, and actin isoform determinations to quantify the relative levels of ASMA in four populations of cultured hair follicle dermal cells, and fibroblasts derived from three regions of adult and comparable areas of 4-d rat skin. Although follicle sheath populations were morphologically similar, they contained variable proportions of cells that expressed ASMA. Tissue from the most positive region in situ, the lower/mid sheath, also gave rise to the most positive cells in culture (98%), followed by the end bulb (85%) and then upper sheath (50%). The follicle dermal cells (including papilla 81%) displayed and maintained levels of expression well above those obtained for adult (below 10%) or 4-d (9-40%) fibroblasts, and even cultured smooth muscle cells. It was also confirmed that levels of expression in adult fibroblasts could be positively correlated with hair follicle density in the biopsies from which they were initiated. Differential expression of ASMA in follicle subpopulations provides an insight into how their behavior may be linked to their specialized functions, for example, their likely involvement in the mechanics of the hair cycle. Moreover, the proposition that hair follicle dermal cells represent unappreciated constituents of general skin fibroblast cultures has substantial implications.