A key role exists for prostaglandins (PGs) in reproductive health, including fertility and parturition. However, the cellular sources and regulation of PG production by cyclooxygenase (COX) in the human female reproductive tract remain poorly understood. We recently reported that human female reproductive tract fibroblasts are divisible into distinct subsets based on their Thy-1 surface expression. Herein, we demonstrate that the expression, induction, and subcellular localization of COX-1 and COX-2 and the downstream PG biosynthesis are markedly different between these subsets. Specifically, Thy-1(+) fibroblasts highly express COX-1, which is responsible for high-level PGE(2) production, a feature usually attributed to the COX-2 isoenzyme. In contrast, COX-2, generally considered an inducible isoform, is constitutively expressed in the Thy-1(-) subset, which only minimally produces PGE(2). The intracellular signaling pathways for COX regulation also differ between the subsets. Determination of differences in signal transduction, COX expression and localization, and PG production by human reproductive fibroblast subtypes supports the concept of fibroblast heterogeneity and the possibility that these subsets may play unique roles in tissue homeostasis and in inflammation.