Troy is a TNFR superfamily member that is highly expressed in developing hair follicles. Its possible function and ligand in skin have, however, been unknown. Here we demonstrate that an immunomodulatory cytokine, lymphotoxin-alpha (LTalpha), is a functional ligand of Troy by 3 biochemical approaches: (1) Immunoprecipitation assays revealed that LTalpha, but not LTbeta or any obligate combination of LTalpha and LTbeta, binds to Troy. (2) Co-transfection of LTalpha with Troy sharply upregulated NFkappaB reporter transcription, whereas LTbeta or a combination of LTalpha and LTbeta did not. (3) Recombinant LTalpha protein upregulated NFkappaB activity through Troy in a dosedependent manner. We further found that LTalpha is expressed in dermal papillae of developing hair follicles, whereas Troy was expressed in adjacent matrix region. This suggested involvement of LTalpha-Troy signaling in mesenchyme-epithelium interactions during hair follicle development. However, in Troy mutant mice that we generated, hair subtype composition and morphology were altered slightly if at all. The present study thus suggested a subtle function of the newly identified LTalpha-Troy pathway in skin appendage development, however, it may have an additional action compensated by a parallel EDA signaling pathway.