Little is known about fibroblasts from the female reproductive tract, much less whether or not functional subsets exist. Fibroblasts are key as sentinel cells for recruiting white blood cells and for wound healing. The purpose of this research was to evaluate the possibility that functional subsets of fibroblasts exist in the human female reproductive tract. The strategy used was to define fibroblast subpopulations based on their surface expression of the Thy 1 antigen. In situ staining of human myometrium and endometrium showed heterogeneous staining for Thy 1. Freshly derived strains of fibroblasts from the myometrium and endometrium also demonstrated heterogeneous Thy 1 expression. For the first time, using magnetic beading and fluorescence-activated cell sorting, human myometrial fibroblasts were successfully separated into functionally unique Thy 1⁺ and Thy 1⁻ subsets. Both subsets produced the proinflammatory cytokines interleukin (IL)-6 and IL-8 after IL-1β stimulation, but only the Thy 1⁺ subset produced MCP-1. Furthermore, only Thy 1⁺ fibroblasts up-regulated CD40 surface expression with IL-1β or interferon-γ treatment. Engagement of CD40 in the Thy 1⁺ subpopulation induced IL-6, IL-8, and MCP-1. The discovery of functional subsets of reproductive tract fibroblasts now permits assessment of their roles in the normal functions of the reproductive tract and in disease states such as adhesions and menorrhagia.